AXMI-007, a delta-endotoxin gene and methods for its use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for a delta-endotoxin polypeptide are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated delta-endotoxin nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for isolated nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences shown in SEQ ID NOS:2 and 4 and the nucleotide sequences set forth in SEQ ID NOS:1 and 3, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/448,812, filed Feb. 20, 2003, the contents ofwhich are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] This invention relates to the field of molecular biology.Provided are novel genes that encode pesticidal proteins. These proteinsand the nucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

[0003]Bacillus thuringiensis is a Gram-positive spore forming soilbacterium characterized by its ability to produce crystalline inclusionsthat are specifically toxic to certain orders and species of insects,but are harmless to plants and other non-targeted organisms. For thisreason, compositions including Bacillus thuringiensis strains or theirinsecticidal proteins can be used as environmentally acceptableinsecticides to control agricultural insect pests or insect vectors fora variety of human or animal diseases.

[0004] Crystal (Cry) proteins (delta-endotoxins) from Bacillusthuringiensis have potent insecticidal activity against predominantlyLepidopteran, Dipteran, and Coleopteran larvae. These proteins also haveshown activity against Hymenoptera, Homoptera, Phthiraptera, Mallophaga,and Acari pest orders, as well as other invertebrate orders such asNemathelminthes, Platyhelminthes, and Sarcomastigorphora (Feitelson(1993) The Bacillus Thuringiensis family tree. In Advanced EngineeredPesticides. Marcel Dekker, Inc., New York, N.Y.) These proteins wereoriginally classified as CryI to CryV based primarily on theirinsecticidal activity. The major classes were Lepidoptera-specific (I),Lepidoptera- and Diptera-specific (II), Coleoptera-specific (III),Diptera-specific (IV), and nematode-specific (V) and (VI). The proteinswere further classified into subfamilies; more highly related proteinswithin each family were assigned divisional letters such as Cry1A,Cry1B, Cry1C, etc. Even more closely related proteins within eachdivision were given names such as Cry1C1, Cry1C2, etc.

[0005] A new nomenclature was recently described for the Cry genes basedupon amino acid sequence homology rather than insect target specificity(Crickmore et al. (1998) Microbiol. Mol. Biol. Rev. 62:807-813). In thenew classification, each toxin is assigned a unique name incorporating aprimary rank (an Arabic number), a secondary rank (an uppercase letter),a tertiary rank (a lowercase letter), and a quaternary rank (anotherArabic number). In the new classification, Roman numerals have beenexchanged for Arabic numerals in the primary rank. Proteins with lessthan 45% sequence identity have different primary ranks, and thecriteria for secondary and tertiary ranks are 78% and 95%, respectively.

[0006] The crystal protein does not exhibit insecticidal activity untilit has been ingested and solubilized in the insect midgut. The ingestedprotoxin is hydrolyzed by proteases in the insect digestive tract to anactive toxic molecule. (Hofte and Whiteley (1989) Microbiol. Rev.53:242-255). This toxin binds to apical brush border receptors in themidgut of the target larvae and inserts into the apical membranecreating ion channels or pores, resulting in larval death.

[0007] Delta-endotoxins generally have five conserved sequence domains,and three conserved structural domains (see, for example, de Maagd etal. (2001) Trends Genetics 17:193-199). The first conserved structuraldomain consists of seven alpha helices and is involved in membraneinsertion and pore formation. Domain II consists of three beta-sheetsarranged in a Greek key configuration, and domain III consists of twoantiparallel beta-sheets in ‘jelly-roll’ formation (de Maagd et al.(2001) supra). Domains II and III are involved in receptor recognitionand binding, and are therefore considered determinants of toxinspecificity.

[0008] Because of the devastation that insects can confer, there is acontinual need to discover new forms of Bacillus thuringiensisdelta-endotoxins.

SUMMARY OF INVENTION

[0009] Compositions and methods for conferring pesticide resistance tobacteria, plants, plant cells, tissues, and seeds are provided.Compositions include isolated nucleic acid molecules encoding sequencesfor delta-endotoxin polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude isolated or recombinant polypeptide sequences of the endotoxin,compositions comprising these polypeptides, and antibodies to thosepolypeptides. The nucleotide sequences can be used in DNA constructs orexpression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed for optimumexpression in an organism, including, but not limited to, amicroorganism or a plant. Compositions also comprise transformedbacteria, plants, plant cells, tissues, and seeds.

[0010] In particular, the present invention provides for an isolatednucleic acid molecule comprising the nucleotide sequences encoding theamino acid sequences shown in SEQ ID NOS:2 and 4 and the nucleotidesequences set forth in SEQ ID NOS:1 and 3, as well as variants andfragments thereof. Nucleotide sequences that are complementary to anucleotide sequence of the invention, or that hybridize to a sequence ofthe invention, are also encompassed.

[0011] Methods are provided for producing the polypeptides of theinvention, and for using those polypeptides for controlling or killing alepidopteran or coleopteran pest.

[0012] The compositions and methods of the invention are useful for theproduction of organisms with pesticide resistance, specifically bacteriaand plants. These organisms and compositions derived from them aredesirable for agricultural purposes. The compositions of the inventionare also useful for generating altered or improved delta-endotoxinproteins that have pesticidal activity, or for detecting the presence ofdelta-endotoxin proteins or nucleic acids in products or organisms.

DESCRIPTION OF FIGURES

[0013]FIGS. 1A, B, and C show an alignment of AXMI-007 (SEQ ID NO:2)with cry1Aa (SEQ ID NO:5), cry1Ac (SEQ ID NO:6), cry1Ia (SEQ ID NO:7),cry3Aa1 (SEQ ID NO:8), cry3Ba (SEQ ID NO:9), cry4Aa (SEQ ID NO:10),cry6Aa (SEQ ID NO:11), cry7Aa (SEQ ID NO:12), cry8Aa (SEQ ID NO:13),cry10Aa (SEQ ID NO:14), cry16Aa (SEQ ID NO:15), cry19Ba (SEQ ID NO:16),and cry24Aa (SEQ ID NO:17). Toxins having C-terminal non-toxic domainswere artificially truncated as shown. The alignment shows the mosthighly conserved amino acid residues highlighted in black, and highlyconserved amino acid residues highlighted in gray. Conserved group 1 isfound from about amino acid residue 217 to about 238 of SEQ ID NO:2.Conserved group 2 is found from about amino acid residue 299 to about347 of SEQ ID NO:2. Conserved group 3 is found from about amino acidresidue 445 to about 590 of SEQ ID NO:2. Conserved group 4 is found fromabout amino acid residue 609 to about 619 of SEQ ID NO:2. Conservedgroup 5 is found from about amino acid residue 692 to about 702 of SEQID NO:2.

DETAILED DESCRIPTION

[0014] The present invention is drawn to compositions and methods forregulating pest resistance in organisms, particularly plants or plantcells. The methods involve transforming organisms with a nucleotidesequence encoding a delta-endotoxin protein of the invention. Inparticular, the nucleotide sequences of the invention are useful forpreparing plants and microorganisms that possess pesticidal activity.Thus, transformed bacteria, plants, plant cells, plant tissues and seedsare provided. Compositions include delta-endotoxin nucleic acids andproteins of Bacillus thuringiensis. The sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of otherdelta-endotoxin genes, and for the generation of altered pesticidalproteins by methods known in the art, such as domain swapping or DNAshuffling. The proteins find use in controlling or killing lepidopteranor coleopteran pest populations and for producing compositions withpesticidal activity.

[0015] Definitions

[0016] By “delta-endotoxin” is intended a toxin from Bacillusthuringiensis that has toxic activity against one or more pests,including, but not limited to, members of the Lepidoptera, Diptera, andColeoptera orders. In some cases, delta-endotoxin proteins have beenisolated from other organisms, including Clostridium bifermentans andPaenibacillus popilliae. Delta-endotoxin proteins include amino acidsequences deduced from the full-length nucleotide sequences disclosedherein, and amino acid sequences that are shorter than the full-lengthsequences, either due to the use of an alternate downstream start site,or due to processing that produces a shorter protein having pesticidalactivity. Processing may occur in the organism the protein is expressedin, or in the pest after ingestion of the protein. Delta-endotoxinsinclude proteins identified as cry1 through cry43, cyt1 and cyt2, andCyt-like toxin. There are currently over 250 known species ofdelta-endotoxins with a wide range of specificities and toxicities. Foran expansive list see Crickmore et al. (1998), Microbiol. Mol. Biol.Rev. 62:807-813, and for regular updates see Crickmore et al. (2003)“Bacillus thuringiensis toxin nomenclature,” atwww.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.

[0017] Bacterial genes, such as the AXMI-007 gene of this invention,quite often possess multiple methionine initiation codons in proximityto the start of the open reading frame. Often, translation initiation atone or more of these start codons will lead to generation of afunctional protein. These start codons can include ATG codons. However,bacteria such as Bacillus sp. also recognize the codon GTG as a startcodon, and proteins that initiate translation at GTG codons contain amethionine at the first amino acid. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of delta-endotoxinproteins that encode pesticidal activity. For example, an alternatestart site for a delta-endotoxin protein of the invention may be at basepair 151 of SEQ ID NO:1. Translation from this alternate start siteresults in the amino acid sequence found in SEQ ID NO:4. Thesedelta-endotoxin proteins are encompassed in the present invention andmay be used in the methods of the present invention.

[0018] By “plant cell” is intended all known forms of plant, includingundifferentiated tissue (e.g. callus), suspension culture cells,protoplasts, leaf cells, root cells, phloem cells, plant seeds, pollen,propagules, embryos and the like. By “plant expression cassette” isintended a DNA construct that is capable of resulting in the expressionof a protein from an open reading frame in a plant cell. Typically thesecontain a promoter and a coding sequence. Often, such constructs willalso contain a 3′ untranslated region. Such constructs may contain a‘signal sequence’ or ‘leader sequence’ to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum, or Golgi apparatus.

[0019] By “signal sequence” is intended a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. By “leader sequence” is intended any sequence that whentranslated, results in an amino acid sequence sufficient to triggerco-translational transport of the peptide chain to a sub-cellularorganelle. Thus, this includes leader sequences targeting transportand/or glycosylation by passage-into the endoplasmic reticulum, passageto vacuoles, plastids including chloroplasts, mitochondria, and thelike.

[0020] By “plant transformation vector” is intended a DNA molecule thatis necessary for efficient transformation of a plant cell. Such amolecule may consist of one or more plant expression cassettes, and maybe organized into more than one ‘vector’ DNA molecule. For example,binary vectors are plant transformation vectors that utilize twonon-contiguous DNA vectors to encode all requisite cis- and trans-actingfunctions for transformation of plant cells (Hellens and Mullineaux(2000) Trends in Plant Science 5:446-451). “Vector” refers to a nucleicacid construct designed for transfer between different host cells.“Expression vector” refers to a vector that has ability to incorporate,integrate and express heterologous DNA sequences or fragments in aforeign cell.

[0021] “Transgenic plants” or “transformed plants” or “stablytransformed plants or cells or tissues” refers to plants that haveincorporated or integrated exogenous nucleic acid sequences or DNAfragments into the plant cell. These nucleic acid sequences includethose that are exogenous, or not present in the untransformed plantcell, as well as those that may be endogenous, or present in theuntransformed plant cell. “Heterologous” generally refers to the nucleicacid sequences that are not endogenous to the cell or part of the nativegenome in which they are present, and have been added to the cell byinfection, transfection, microinjection, electroporation,microprojection, or the like.

[0022] “Promoter” refers to a nucleic acid sequence that functions todirect transcription of a downstream coding sequence. The promotertogether with other transcriptional and translational regulatory nucleicacid sequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

[0023] Provided herein are novel isolated nucleotide sequences thatconfer pesticidal activity. Also provided are the amino acid sequencesfor the delta-endotoxin proteins. The protein resulting from translationof this gene allows cells to control or kill pests that ingest it.

[0024] An “isolated” or “purified” nucleic acid molecule or protein, orbiologically active portion thereof, is substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized. Preferably, an “isolated” nucleicacid is free of sequences (preferably protein encoding sequences) thatnaturally flank the nucleic acid (i.e., sequences located at the 5′ and3′ ends of the nucleic acid) in the genomic DNA of the organism fromwhich the nucleic acid is derived. For purposes of the invention,“isolated” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, the isolateddelta-endotoxin-encoding nucleic acid molecule can contain less thanabout 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotidesequence that naturally flanks the nucleic acid molecule in genomic DNAof the cell from which the nucleic acid is derived. A delta-endotoxinprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, or 5% (bydry weight) of non-delta-endotoxin protein (also referred to herein as a“contaminating protein”). Various aspects of the invention are describedin further detail in the following subsections.

[0025] Isolated Nucleic Acid Molecules, and Variants and FragmentsThereof

[0026] One aspect of the invention pertains to isolated nucleic acidmolecules comprising nucleotide sequences encoding delta-endotoxinproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify delta-endotoxin encoding nucleic acids. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA.

[0027] Nucleotide sequences encoding the proteins of the presentinvention include the sequences set forth in SEQ ID NOS:1 and 3, andcomplements thereof. By “complement” is intended a nucleotide sequencethat is sufficiently complementary to a given nucleotide sequence suchthat it can hybridize to the given nucleotide sequence to thereby form astable duplex. The corresponding amino acid sequences for thedelta-endotoxin proteins encoded by these nucleotide sequences are setforth in SEQ ID NOS:2 and 4.

[0028] Nucleic acid molecules that are fragments of thesedelta-endotoxin encoding nucleotide sequences are also encompassed bythe present invention. By “fragment” is intended a portion of thenucleotide sequence encoding a delta-endotoxin protein. A fragment of anucleotide sequence may encode a biologically active portion of adelta-endotoxin protein, or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Nucleicacid molecules that are fragments of a delta-endotoxin nucleotidesequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350,400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050,1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650,1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200nucleotides, or up to the number of nucleotides present in a full-lengthdelta-endotoxin encoding nucleotide sequence disclosed herein (forexample, 2235 nucleotides for SEQ ID NO:1, or 2085 nucleotides for SEQID NO:3) depending upon the intended use. Fragments of the nucleotidesequences of the present invention will encode protein fragments thatretain the biological activity of the delta-endotoxin protein and,hence, retain pesticidal activity. By “retains activity” is intendedthat the fragment will have at least about 30%, preferably at leastabout 50%, more preferably at least about 70%, even more preferably atleast about 80% of the pesticidal activity of the delta-endotoxinprotein. Methods for measuring pesticidal activity are well known in theart. See, for example, Czapla and Lang (1990) J. Econ. Entomol. 83(6):2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone et al.(1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

[0029] A fragment of a delta-endotoxin encoding nucleotide sequence thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, 600, 650, or 700 contiguous amino acids,or up to the total number of amino acids present in a full-lengthdelta-endotoxin protein of the invention (for example, 744 amino acidsfor SEQ ID NO:2 or 694 for SEQ ID NO:4).

[0030] Preferred delta-endotoxin proteins of the present invention areencoded by a nucleotide sequence sufficiently identical to thenucleotide sequence of SEQ ID NO:1 or 3. By “sufficiently identical” isintended an amino acid or nucleotide sequence that has at least about60% or 65% sequence identity, preferably about 70% or 75% sequenceidentity, more preferably about 80% or 85% sequence identity, mostpreferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity compared to a reference sequence using one of thealignment programs described herein using standard parameters. One ofskill in the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like.

[0031] To determine the percent identity of two amino acid sequences orof two nucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

[0032] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm. A nonlimiting example ofa mathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous todelta-endotoxin nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous todelta-endotoxin protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See, Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Another preferred, non-limiting exampleof a mathematical algorithm utilized for the comparison of sequences isthe ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res.22:4673-4680). ClustalW compares sequences and aligns the entirety ofthe amino acid or DNA sequence, and thus can provide data about thesequence conservation of the entire amino acid sequence. The ClustalWalgorithm is used in several commercially available DNA/amino acidanalysis software packages, such as the ALIGNX module of the vector NTiProgram Suite (Informax, Inc). After alignment of amino acid sequenceswith ClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GeneDoc™. Genedoc™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. See, www.ncbi.nlm.nih.gov. Another non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0),which is part of the GCG sequence alignment software package (availablefrom Accelrys, Inc., 9865 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

[0033] A preferred program is GAP version 10, which used the algorithmof Needleman and Wunsch (1970) supra. GAP Version 10 may be used withthe following parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 Scoring Matrix. Equivalent programs may also be used. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

[0034] The invention also encompasses variant nucleic acid molecules.“Variants” of the delta-endotoxin-encoding nucleotide sequences includethose sequences that encode the delta-endotoxin proteins disclosedherein but that differ conservatively because of the degeneracy of thegenetic code as well as those that are sufficiently identical asdiscussed above. Naturally occurring allelic variants can be identifiedwith the use of well-known molecular biology techniques, such aspolymerase chain reaction (PCR) and hybridization techniques as outlinedbelow. Variant nucleotide sequences also include synthetically derivednucleotide sequences that have been generated, for example, by usingsite-directed mutagenesis but which still encode the delta-endotoxinproteins disclosed in the present invention as discussed below. Variantproteins encompassed by the present invention are biologically active,that is they continue to possess the desired biological activity of thenative protein, that is, retaining pesticidal activity. By “retainsactivity” is intended that the variant will have at least about 30%,preferably at least about 50%, more preferably at least about 70%, evenmore preferably at least about 80% of the pesticidal activity of thenative protein. Methods for measuring pesticidal activity are well knownin the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83(6): 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marroneet al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

[0035] The skilled artisan will further appreciate that changes can beintroduced by mutation into the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodeddelta-endotoxin proteins, without altering the biological activity ofthe proteins. Thus, variant isolated nucleic acid molecules can becreated by introducing one or more nucleotide substitutions, additions,or deletions into the corresponding nucleotide sequence disclosedherein, such that one or more amino acid substitutions, additions ordeletions are introduced into the encoded protein. Mutations can beintroduced by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Such variant nucleotide sequences are alsoencompassed by the present invention.

[0036] For example, preferably, conservative amino acid substitutionsmay be made at one or more predicted, preferably nonessential amino acidresidues. A “nonessential” amino acid residue is a residue that can bealtered from the wild-type sequence of a delta-endotoxin protein withoutaltering the biological activity, whereas an “essential” amino acidresidue is required for biological activity. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art. Thesefamilies include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Such substitutions would not bemade for conserved amino acid residues, or for amino acid residuesresiding within a conserved motif, where such residues are essential forprotein activity.

[0037] There are generally five highly conserved regions among thedelta-endotoxin proteins, concentrated largely in the center of thedomain or at the junction between domains (Rajamohan et al. (1998) Prog.Nucleic Acid Res. Mol. Biol. 60:1-23). The blocks of conserved aminoacids for various delta-endotoxins as well as consensus sequences may befound in Schnepf et al. (1998) Microbio. Mol. Biol. Rev. 62:775-806 andLereclus et al. (1989) Role, Structure, and Molecular Organization ofthe Genes Coding for the Parasporal d-endotoxins of Bacillusthuringiensis. In Regulation of Procaryotic Development. Issar Smit,Slepecky, R. A., Setlow, P. American Society for Microbiology,Washington, D.C. 20006. It has been proposed that delta-endotoxinshaving these conserved regions may share a similar structure, consistingof three domains (Li et al. (1991) Nature 353: 815-821). Domain I hasthe highest similarity between delta-endotoxins (Bravo (1997) J.Bacteriol. 179:2793-2801).

[0038] Amino acid substitutions may be made in nonconserved regions thatretain function. In general, such substitutions would not be made forconserved amino acid residues, or for amino acid residues residingwithin a conserved motif, where such residues are essential for proteinactivity. Examples of residues that are conserved and that may beessential for protein activity include, for example, residues that areidentical between all proteins contained in the alignment of FIGS. 1A,B, and C. Examples of residues that are conserved but that may allowconservative amino acid substitutions and still retain activity include,for example, residues that have only conservative substitutions betweenall proteins contained in the alignment of FIGS. 1A, B, and C. However,one of skill in the art would understand that functional variants mayhave minor conserved or nonconserved alterations in the conservedresidues.

[0039] Alternatively, variant nucleotide sequences can be made byintroducing mutations randomly along all or part of the coding sequence,such as by saturation mutagenesis, and the resultant mutants can bescreened for ability to confer pesticidal activity to identify mutantsthat retain activity. Following mutagenesis, the encoded protein can beexpressed recombinantly, and the activity of the protein can bedetermined using standard assay techniques.

[0040] Using methods such as PCR, hybridization, and the likecorresponding delta-endotoxin sequences can be identified, suchsequences having substantial identity to the sequences of the invention.See, for example, Sambrook J., and Russell, D. W. (2001) MolecularCloning: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide toMethods and Applications (Academic Press, NY).

[0041] In a hybridization method, all or part of the delta-endotoxinnucleotide sequence can be used to screen cDNA or genomic libraries.Methods for construction of such cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook and Russell,2001. The so-called hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker, such as other radioisotopes, a fluorescent compound, an enzyme,or an enzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known delta-endotoxin encodingnucleotide sequence disclosed herein. Degenerate primers designed on thebasis of conserved nucleotides or amino acid residues in the nucleotidesequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleotide sequence thathybridizes under stringent conditions to at least about 12, preferablyabout 25, more preferably at least about 50, 75, 100, 125, 150, 175,200, 250, 300, 350, or 400 consecutive nucleotides ofdelta-endotoxin-encoding nucleotide sequence of the invention or afragment or variant thereof. Preparation of probes for hybridization isgenerally known in the art and is disclosed in Sambrook and Russell,2001, herein incorporated by reference.

[0042] In hybridization techniques, all or part of a known nucleotidesequence is used as a probe that selectively hybridizes to othercorresponding nucleotide sequences present in a population of clonedgenomic DNA fragments or cDNA fragments (i.e., genomic or cDNAlibraries) from a chosen organism. The hybridization probes may begenomic DNA fragments, cDNA fragments, RNA fragments, or otheroligonucleotides, and may be labeled with a detectable group such as³²P, or any other detectable marker. Thus, for example, probes forhybridization can be made by labeling synthetic oligonucleotides basedon the delta-endotoxin sequence of the invention. Methods forpreparation of probes for hybridization and for construction of cDNA andgenomic libraries are generally known in the art and are disclosed inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0043] For example, the entire delta-endotoxin sequence disclosedherein, or one or more portions thereof, may be used as a probe capableof specifically hybridizing to corresponding delta-endotoxin-likesequences and messenger RNAs. To achieve specific hybridization under avariety of conditions, such probes include sequences that are unique andare preferably at least about 10 nucleotides in length, and mostpreferably at least about 20 nucleotides in length. Such probes may beused to amplify corresponding delta-endotoxin sequences from a chosenorganism by PCR. This technique may be used to isolate additional codingsequences from a desired organism or as a diagnostic assay to determinethe presence of coding sequences in an organism. Hybridizationtechniques include hybridization screening of plated DNA libraries(either plaques or colonies; see, for example, Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

[0044] Hybridization of such sequences may be carried out understringent conditions. By “stringent conditions” or “stringenthybridization conditions” is intended conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

[0045] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1×to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5×to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

[0046] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl (1984) Anal.Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (%form)-500/L; where M is the molarity of monovalent cations, % GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridization,and/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with >90% identity aresought, the T_(m) can be decreased 110° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.). Isolated Proteins and Variants and FragmentsThereof Delta-endotoxin proteins are also encompassed within the presentinvention. By “delta-endotoxin protein” is intended a protein having theamino acid sequence set forth in SEQ ID NO:2 or 4. Fragments,biologically active portions, and variants thereof are also provided,and may be used to practice the methods of the present invention.

[0047] “Fragments” or “biologically active portions” include polypeptidefragments comprising a portion of an amino acid sequence encoding adelta-endotoxin protein as set forth in SEQ ID NO:2 or 4 and that retainpesticidal activity. A biologically active portion of a delta-endotoxinprotein can be a polypeptide which is, for example, 10, 25, 50, 100 ormore amino acids in length. Such biologically active portions can beprepared by recombinant techniques and evaluated for pesticidalactivity. Methods for measuring pesticidal activity are well known inthe art. See, for example, Czapla and Lang (1990) J Econ. Entomol.83(6): 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marroneet al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. As used here, a fragment comprises at least 8 contiguous aminoacids of SEQ ID NO:2 or 4. The invention encompasses other fragments,however, such as any fragment in the protein greater than about 10, 20,30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 and700 amino acids.

[0048] By “variants” is intended proteins or polypeptides having anamino acid sequence that is at least about 60%, 65%, preferably about70%, 75%, more preferably about 80%, 85%, most preferably about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence of SEQ ID NO:2 or 4. Variants also include polypeptidesencoded by a nucleic acid molecule that hybridizes to the nucleic acidmolecule of SEQ ID NO:1 or 3, or a complement thereof, under stringentconditions. Such variants generally retain pesticidal activity. Variantsinclude polypeptides that differ in amino acid sequence due tomutagenesis. Variant proteins encompassed by the present invention arebiologically active, that is they continue to possess the desiredbiological activity of the native protein, that is, retaining pesticidalactivity. Methods for measuring pesticidal activity are well known inthe art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83(6): 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marroneet al. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

[0049] Altered or Improved Variants

[0050] It is recognized that DNA sequences of a delta-endotoxin may bealtered by various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by the delta-endotoxin of the present invention. Thisprotein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the delta-endotoxin protein can be prepared bymutations in the DNA. This may also be accomplished by one of severalforms of mutagenesis and/or in directed evolution. In some aspects, thechanges encoded in the amino acid sequence will not substantially affectthe function of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability ofdelta-endotoxin to confer pesticidal activity may be improved by the useof such techniques upon the compositions of this invention. For example,one may express delta-endotoxin in host cells that exhibit high rates ofbase misincorporation during DNA replication, such as XL-1 Red(Stratagene). After propagation in such strains, one can isolate thedelta-endotoxin DNA (for example by preparing plasmid DNA, or byamplifying by PCR and cloning the resulting PCR fragment into a vector),culture the delta-endotoxin mutations in a non-mutagenic strain, andidentify mutated delta-endotoxin genes with pesticidal activity, forexample by performing an assay to test for pesticidal activity.Generally, the protein is mixed and used in feeding assays. See, forexample Marrone et al. (1985) J. of Economic Entomology 78:290-293. Suchassays can include contacting plants with one or more pests anddetermining the plant's ability to survive and/or cause the death of thepests. Examples of mutations that result in increased toxicity are foundin Schnepf et al. (1998) Microbiol. Mol. Biol. Rev. 62:775-806.

[0051] Alternatively, alterations may be made to the protein sequence ofmany proteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

[0052] Variant nucleotide and amino acid sequences of the presentinvention also encompass sequences derived from mutagenic andrecombinogenic procedures such as DNA shuffling. With such a procedure,one or more different delta-endotoxin protein coding regions can be usedto create a new delta-endotoxin protein possessing the desiredproperties. In this manner, libraries of recombinant polynucleotides aregenerated from a population of related sequence polynucleotidescomprising sequence regions that have substantial sequence identity andcan be homologously recombined in vitro or in vivo. For example, usingthis approach, sequence motifs encoding a domain of interest may beshuffled between the delta-endotoxin gene of the invention and otherknown delta-endotoxin genes to obtain a new gene coding for a proteinwith an improved property of interest, such as an increased insecticidalactivity. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0053] Domain swapping or shuffling is another mechanism for generatingaltered delta-endotoxin proteins. Domains II and III may be swappedbetween delta-endotoxin proteins, resulting in hybrid or chimeric toxinswith improved pesticidal activity or target spectrum. Methods forgenerating recombinant proteins and testing them for pesticidal activityare well known in the art (see, for example, Naimov et al. (2001) Appl.Environ. Microbiol. 67:5328-5330; de Maagd et al. (1996) Appl. Environ.Microbiol. 62:1537-1543; Ge et al. (1991) J. Biol. Chem.266:17954-17958; Schnepf et al. (1990) J. Biol. Chem. 265:20923-20930;Rang et al. 91999) Appl. Environ. Micriobiol. 65:2918-2925).

[0054] Plant Transformation

[0055] Transformation of plant cells can be accomplished by one ofseveral techniques known in the art. First, one engineers thedelta-endotoxin gene in a way that allows its expression in plant cells.Typically a construct that expresses such a protein would contain apromoter to drive transcription of the gene, as well as a 3′untranslated region to allow transcription termination andpolyadenylation. The organization of such constructs is well known inthe art. In some instances, it may be useful to engineer the gene suchthat the resulting peptide is secreted, or otherwise targeted within theplant cell. For example, the gene can be engineered to contain a signalpeptide to facilitate transfer of the peptide to the endoplasmicreticulum. It may also be preferable to engineer the plant expressioncassette to contain an intron, such that mRNA processing of the intronis required for expression.

[0056] Typically this ‘plant expression cassette’ will be inserted intoa ‘plant transformation vector’. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas ‘binary vectors’. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a ‘gene of interest’ (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the gene of interest are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as in understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science, 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethelene glycol, etc.

[0057] In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6: 271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plantlets are found inAyres and Park, 1994 (Critical Reviews in Plant Science 13: 219-239) andBommineni and Jauhar, 1997 (Maydica 42: 107-120). Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

[0058] Generation of transgenic plants may be performed by one ofseveral methods, including but not limited to introduction ofheterologous DNA by Agrobacterium into plant cells(Agrobacterium-mediated transformation), bombardment of plant cells withheterologous foreign DNA adhered to particles, and various othernon-particle direct-mediated methods (e.g. Hiei et al. (1994) The PlantJournal 6: 271-282; Ishida et al. (1996) Nature Biotechnology 14:745-750; Ayres and Park (1994) Critical Reviews in Plant Science 13:219-239; Bommineni and Jauhar (1997) Maydica 42: 107-120) to transferDNA.

[0059] Transformation protocols as well as protocols for introducingnucleotide sequences into plants may vary depending on the type of plantor plant cell, i.e., monocot or dicot, targeted for transformation.Suitable methods of introducing nucleotide sequences into plant cellsand subsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (U.S. Pat. No. 5,563,055; U.S.Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBOJ. 3:2717-2722), and ballistic particle acceleration (see, for example,U.S. Pat. No. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No.5,886,244; U.S. Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNATransfer into Intact Plant Cells via Microprojectile Bombardment,” inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborgand Phillips (Springer-Verlag, Berlin); McCabe et al. (1988)Biotechnology 6:923-926); aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066);and Lecl transformation (WO 00/28058). Also see Weissinger et al. (1988)Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Scienceand Technology 5:27-37; Christou et al. (1988) Plant Physiol.87:671-674; McCabe et al. (1988) Bio/Technology 6:923-926; Finer andMcMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182; Singh et al.(1998) Theor. Appl. Genet. 96:319-324; Datta et al. (1990) Biotechnology8:736-740; Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309;U.S. Pat. No. 5,240,855; U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomeset al. (1995) “Direct DNA Transfer into Intact Plant Cells viaMicroprojectile Bombardment,” in Plant Cell, Tissue, and Organ Culture:Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize);Klein et al. (1988) Plant Physiol. 91:440-444; Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369;Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209;Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al.(1992) Theor. Appl. Genet. 84:560-566; D'Halluin et al. (1992) PlantCell 4:1495-1505; Li et al. (1993) Plant Cell Reports 12:250-255 andChristou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda etal. (1996) Nature Biotechnology 14:745-750; all of which are hereinincorporated by reference.

[0060] Following integration of heterologous foreign DNA into plantcells, one then applies a maximum threshold level of appropriateselection in the medium to kill the untransformed cells and separate andproliferate the putatively transformed cells that survive from thisselection treatment by transferring regularly to a fresh medium. Bycontinuous passage and challenge with appropriate selection, oneidentifies and proliferates the cells that are transformed with theplasmid vector. Then molecular and biochemical methods will be used forconfirming the presence of the integrated heterologous gene of interestin the genome of transgenic plant.

[0061] The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

[0062] The delta-endotoxin sequences of the invention may be provided inexpression cassettes for expression in the plant of interest. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

[0063] Such an expression cassette is provided with a plurality ofrestriction sites for insertion of the delta-endotoxin sequence to beunder the transcriptional regulation of the regulatory regions.

[0064] The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and atranscriptional and translational termination region (i.e., terminationregion) functional in plants. The promoter may be native or analogous,or foreign or heterologous, to the plant host and/or to the DNA sequenceof the invention. Additionally, the promoter may be the natural sequenceor alternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

[0065] The termination region may be native with the transcriptionalinitiation region, may be native with the operably-linked DNA sequenceof interest, may be native with the plant host, or may be derived fromanother source (i.e., foreign or heterologous to the promoter, the DNAsequence of interest, the plant host, or any combination thereof).Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also Guerineau et al. (1991) Mol. Gen. Genet.262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991)Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroeet al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res.17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0066] Where appropriate, the gene(s) may be optimized for increasedexpression in the transformed host cell. That is, the genes can besynthesized using host cell-preferred codons for improved expression, ormay be synthesized using codons at a host-preferred codon usagefrequency. Generally, the GC content of the gene will be increased. See,for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for adiscussion of host-preferred codon usage. Methods are known in the artfor synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.6,320,100; 6,075,185; 5,380,831; and 5,436,391, U.S. PublishedApplication Nos. 20040005600 and 20010003849, and Murray et al. (1989)Nucleic Acids Res. 17:477-498, herein incorporated by reference.

[0067] In one embodiment, the nucleic acids of interest are targeted tothe chloroplast for expression. In this manner, where the nucleic acidof interest is not directly inserted into the chloroplast, theexpression cassette will additionally contain a nucleic acid encoding atransit peptide to direct the gene product of interest to thechloroplasts. Such transit peptides are known in the art. See, forexample, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clarket al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987)Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res.Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.

[0068] Methods for transformation of chloroplasts are known in the art.See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relieson particle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride et al. (1994)Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0069] The nucleic acids of interest to be targeted to the chloroplastmay be optimized for expression in the chloroplast to account fordifferences in codon usage between the plant nucleus and this organelle.In this manner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

[0070] Evaluation of Plant Transformation

[0071] Following introduction of heterologous foreign DNA into plantcells, the transformation or integration of heterologous gene in theplant genome is confirmed by various methods such as analysis of nucleicacids, proteins and metabolites associated with the integrated gene.

[0072] PCR Analysis: PCR analysis is a rapid method to screentransformed cells, tissue or shoots for the presence of incorporatedgene at the earlier stage before transplanting into the soil (Sambrookand Russell, 2001). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

[0073] Southern Analysis: Plant transformation is confirmed by Southernblot analysis of genomic DNA (Sambrook and Russell, 2001). In general,total DNA is extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” then is probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene in the plant genome according to standardtechniques (Sambrook and Russell, 2001. Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0074] Northern Analysis: RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, blotted onto anylon filter according to standard procedures that are routinely used inthe art (Sambrook, J., and Russell, D. W. 2001. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.). Expression of RNA encoded by the delta-endotoxin is thentested by hybridizing the filter to a radioactive probe derived from adelta-endotoxin, by methods known in the art (Sambrook and Russell,2001).

[0075] Western blot and Biochemical assays: Western blot and biochemicalassays and the like may be carried out on the transgenic plants toconfirm the determine the presence of protein encoded by thedelta-endotoxin gene by standard procedures (Sambrook, J., and Russell,D. W. 2001. Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) using antibodies that bindto one or more epitopes present on the delta-endotoxin protein.

[0076] Pesticidal Activity in Plants

[0077] In another aspect of the invention, one may generate transgenicplants expressing delta-endotoxin that have pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, aerosol beam, biolistictransformation, and non-particle-mediated methods may be used at thediscretion of the experimenter. Plants expressing delta-endotoxin may beisolated by common methods described in the art, for example bytransformation of callus, selection of transformed callus, andregeneration of fertile plants from such transgenic callus. In suchprocess, one may use any gene as a selectable marker so long as itsexpression in plant cells confers ability to identify or select fortransformed cells.

[0078] A number of markers have been developed for use with plant cells,such as resistance to chloramphenicol, the aminoglycoside G418,hygromycin, or the like. Other genes that encode a product involved inchloroplast metabolism may also be used as selectable markers. Forexample, genes that provide resistance to plant herbicides such asglyphosate, bromoxynil, or imidazolinone may find particular use. Suchgenes have been reported (Stalker et al. (1985) J. Biol. Chem.263:6310-6314 (bromoxynil resistance nitrilase gene); and Sathasivan etal. (1990) Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistancegene).

[0079] Fertile plants expressing delta-endotoxin may be tested forpesticidal activity, and the plants showing optimal activity selectedfor further breeding. Methods are available in the art to assay for pestactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293.

[0080] Use in Pesticidal Control

[0081] General methods for employing the strains of the invention inpesticide control or in engineering other organisms as pesticidal agentsare known in the art. See, for example U.S. Pat. No. 5,039,523 and EP0480762A2.

[0082] The Bacillus strains of the invention or the microorganisms whichhave been genetically altered to contain the pesticidal gene and proteinmay be used for protecting agricultural crops and products from pests.In one aspect of the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

[0083] Alternatively, the pesticide is produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. In one aspect of this invention, thesecells are then treated under conditions that prolong the activity of thetoxin produced in the cell when the cell is applied to the environmentof target pest(s). The resulting product retains the toxicity of thetoxin. These naturally encapsulated pesticides may then be formulated inaccordance with conventional techniques for application to theenvironment hosting a target pest, e.g., soil, water, and foliage ofplants. See, for example EPA 0192319, and the references cited therein.Alternatively, one may formulate the cells expressing the genes of thisinvention such as to allow application of the resulting material as apesticide.

[0084] The active ingredients of the present invention are normallyapplied in the form of compositions and can be applied to the crop areaor plant to be treated, simultaneously or in succession, with othercompounds. These compounds can be fertilizers, weed killers,cryoprotectants, surfactants, detergents, pesticidal soaps, dormantoils, polymers, and/or time-release or biodegradable carrierformulations that permit long-term dosing of a target area following asingle application of the formulation. They can also be selectiveherbicides, chemical insecticides, virucides, microbicides, amoebicides,pesticides, fungicides, bacteriocides, nematocides, mollusocides ormixtures of several of these preparations, if desired, together withfurther agriculturally acceptable carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g. natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders or fertilizers.Likewise the formulations may be prepared into edible “baits” orfashioned into pest “traps” to permit feeding or ingestion by a targetpest of the pesticidal formulation.

[0085] Preferred methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention whichcontains at least one of the pesticidal proteins produced by thebacterial strains of the present invention are leaf application, seedcoating and soil application. The number of applications and the rate ofapplication depend on the intensity of infestation by the correspondingpest.

[0086] The composition may be formulated as a powder, dust, pellet,granule, spray, emulsion, colloid, solution, or such like, and may bepreparable by such conventional means as desiccation, lyophilization,homogenation, extraction, filtration, centrifugation, sedimentation, orconcentration of a culture of cells comprising the polypeptide. In allsuch compositions that contain at least one such pesticidal polypeptide,the polypeptide may be present in a concentration of from about 1% toabout 99% by weight.

[0087] Lepidopteran or coleopteran pests may be killed or reduced innumbers in a given area by the methods of the invention, or may beprophylactically applied to an environmental area to prevent infestationby a susceptible pest. Preferably the pest ingests, or is contactedwith, a pesticidally-effective amount of the polypeptide. By“pesticidally-effective amount” is intended an amount of the pesticidethat is able to bring about death to at least one pest, or to noticeablyreduce pest growth, feeding, or normal physiological development. Thisamount will vary depending on such factors as, for example, the specifictarget pests to be controlled, the specific environment, location,plant, crop, or agricultural site to be treated, the environmentalconditions, and the method, rate, concentration, stability, and quantityof application of the pesticidally-effective polypeptide composition.The formulations may also vary with respect to climatic conditions,environmental considerations, and/or frequency of application and/orseverity of pest infestation.

[0088] The pesticide compositions described may be made by formulatingeither the bacterial cell, crystal and/or spore suspension, or isolatedprotein component with the desired agriculturally-acceptable carrier.The compositions may be formulated prior to administration in anappropriate means such as lyophilized, freeze-dried, desiccated, or inan aqueous carrier, medium or suitable diluent, such as saline or otherbuffer. The formulated compositions may be in the form of a dust orgranular material, or a suspension in oil (vegetable or mineral), orwater or oil/water emulsions, or as a wettable powder, or in combinationwith any other carrier material suitable for agricultural application.Suitable agricultural carriers can be solid or liquid and are well knownin the art. The term “agriculturally-acceptable carrier” covers alladjuvants, inert components, dispersants, surfactants, tackifiers,binders, etc. that are ordinarily used in pesticide formulationtechnology; these are well known to those skilled in pesticideformulation. The formulations may be mixed with one or more solid orliquid adjuvants and prepared by various means, e.g., by homogeneouslymixing, blending and/or grinding the pesticidal composition withsuitable adjuvants using conventional formulation techniques. Suitableformulations and application methods are described in U.S. Pat. No.6,468,523, herein incorporated by reference.

[0089] “Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

[0090] Insect pests include insects selected from the orders Coleoptera,Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera,Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera,Trichoptera, etc., particularly Coleoptera and Lepidoptera. Insect pestsof the invention for the major crops include: Maize: Ostrinia nubilalis,European corn borer; Agrotis ipsilon, black cutworm; Helicoverpa zea,corn earworm; Spodoptera frugiperda, fall armyworm; Diatraeagrandiosella, southwestern corn borer; Elasmopalpus lignosellus, lessercornstalk borer; Diatraea saccharalis, surgarcane borer; Diabroticavirgifera, western corn rootworm; Diabrotica longicornis barberi,northern corn rootworm; Diabrotica undecimpunctata howardi, southerncorn rootworm; Melanotus spp., wireworms; Cyclocephala borealis,northern masked chafer (white grub); Cyclocephala immaculata, southernmasked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, two spotted spider mite;Sorghum: Chilopartellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

[0091] Nematodes include parasitic nematodes such as root-knot, cyst,and lesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

[0092] The following examples are offered by way of illustration and notby way of limitation.

EXPERIMENTAL Example 1 Extraction of Plasmid DNA

[0093] A pure culture of strain ATX13026 was grown in large quantitiesof rich media. The culture was spun to harvest the cell pellet. The cellpellet was then prepared by treatment with SDS by methods known in theart, resulting in breakage of the cell wall and release of DNA. Proteinsand large genomic DNA was then precipitated by a high saltconcentration. The plasmid DNA was then precipitated by standard ethanolprecipitation. The plasmid DNA was separated from any remainingchromosomal DNA by high-speed centrifugation through a cesium chloridegradient. The DNA was visualized in the gradient by UV light and theband of lower density (i.e. the lower band) was extracted using asyringe. This band contained the plasmid DNA from Strain ATX 13026. Thequality of the DNA was checked by visualization on an agarose gel bymethods known in the art.

Example 2 Cloning of Genes

[0094] The purified plasmid DNA was sheared into 5-10 kb sized fragmentsand the 5′ and 3′ single stranded overhangs repaired using T4 DNApolymerase and Klenow fragment in the presence of all four dNTPs, asknown in the art. Phosphates were then attached to the 5′ ends bytreatment with T4 polynucleotide kinase, as known in the art. Therepaired DNA fragments were then ligated overnight into a standard highcopy vector (i.e. pBluescript SK+), suitably prepared to accept theinserts as known in the art (for example by digestion with a restrictionenzyme producing blunt ends).

[0095] The quality of the library was analyzed by digesting a subset ofclones with a restriction enzyme known to have a cleavage site flankingthe cloning site. A high percentage of clones were determined to containinserts, with an average insert size of 5-6 kb.

Example 3 High Throughput Sequencing of Library Plates

[0096] Once the shotgun library quality was checked and confirmed,colonies were grown in a rich broth in 2 ml 96-well blocks overnight at37° C. at a shaking speed of 350 rpm. The blocks were spun to harvestthe cells to the bottom of the block. The blocks were then prepared bystandard alkaline lysis prep in a high throughput format.

[0097] The end sequences of clones from this library were thendetermined for a large number of clones from each block in the followingway: The DNA sequence of each clone chosen for analysis was determinedusing the fluorescent dye terminator sequencing technique (AppliedBiosystems) and standard primers flanking each side of the cloning site.Once the reactions had been carried out in the thermocycler, the DNA wasprecipitated using standard ethanol precipitation. The DNA wasresuspended in water and loaded onto a capillary sequencing machine.Each library plate of DNA was sequenced from either end of the cloningsite, yielding two reads per plate over each insert.

Example 4 Assembly and Screening of Sequencing Data

[0098] DNA sequences obtained were compiled into an assembly project andaligned together to form contigs. This can be done efficiently using acomputer program, such as Vector NTi, or alternatively by using thePred/Phrap suite of DNA alignment and analysis programs. These contigs,along with any individual read that may not have been added to a contig,were compared to a compiled database of all classes of known pesticidalgenes. Contigs or individual reads identified as having identity to aknown endotoxin or pesticidal gene were analyzed further. Among thesequences obtained, clone pAX007 contained DNA identified as havinghomology to known endotoxin genes. Therefore, pAX007 was selected forfurther sequencing.

Example 5 Sequencing of pAX007, and Identification of AXMI-007

[0099] Primers were designed to anneal to pAX007, in a manner such thatDNA sequences generated from such primers will overlap existing DNAsequence of the clone(s). This process, known as “oligo walking,” iswell known in the art. This process was utilized to determine the entireDNA sequence of the region exhibiting homology to a known endotoxingene. In the case of pAX007, this process was used to determine the DNAsequence of the entire clone, resulting in a single nucleotide sequence.The completed DNA sequence was then placed back into the original largeassembly for further validation. This allowed incorporation of more DNAsequence reads into the contig, resulting in multiple reads of coverageover the entire region.

[0100] Analysis of the DNA sequence of pAX007 by methods known in theart identified an open reading frame with homology to known deltaendotoxin genes. This open reading frame is designated as AXMI-007. TheDNA sequence of AXMI-007 is provided as SEQ ID NO:1, and the amino acidsequence of the predicted AMXI-007 protein is provided in SEQ ID NO:2.An alternate start site for AXMI-007 at nucleotide 151 of SEQ ID NO:1generates the amino acid sequence provided as SEQ ID NO:4.

Example 6 Homology of AXMI-007 to Known Endotoxin Genes

[0101] Searches of DNA and protein databases with the DNA sequence andamino acid sequence of AXMI-007 reveal that AXMI-007 is homologous toknown endotoxins.

[0102] Blast searches identify cry4Aa as having the strongest block ofhomology, though alignment of AMXI-007 protein (SEQ ID NO:2) to a largeset of endotoxin proteins shows that the most homologous protein iscry10Aa. The overall amino acid identity of cry10Aa to AXMI-007 is 25%(see Table 1). Inspection of the amino acid sequence of AXMI-007suggests that it does not contain a C-terminal non-toxic domain as ispresent in several endotoxin families. By removing this C-terminalprotein of the toxins from the alignment, the alignment reflects theamino acid identify present solely in the toxin domains (see Table 1,column three). This ‘trimmed’ alignment is shown in FIG. 1. TABLE 1Amino Acid Identity of AXMI-007 with Exemplary Endotoxin Classes PercentAmino Acid Percent Amino Acid Identity of Endotoxin Identity to AXMI-007truncated Toxins to AXMI-007 cry1Aa 11% 17% cry1Ac 12% 20% cry1Ia 19%18% cry3Aa 19% 19% cry3Bb 21% 21% cry4Aa 17% 27% cry6Aa 5% 4% cry7Aa 13%19% cry8Aa 13% 20% cry10Aa 25% 25% cry16Aa 24% 24% cry19Ba 25% 25%cry24Aa 19% 19%

Example 7 Homology between AXMI-006 and AXMI-007

[0103] Comparison of the amino acid sequences of AXMI-007 with AXMI-006(see co-pending U.S. Application entitled “AXMI-006, A Delta-EndotoxinGene and Methods For Its Use”, filed concurrently herewith) show thatthe two toxins share significant amino acid homology. Alignment of theamino acid sequence of AXMI-006 and AXMI-007 (SEQ ID NO:2) show theproteins to be 85% identical at the amino acid level. Thus AXMI-006 andAXMI-007 constitute a new class of related endotoxins.

Example 8 Assays for Pesticidal Activity

[0104] The ability of a pesticidal protein to act as a pesticide upon apest is often assessed in a number of ways. One way well known in theart is to perform a feeding assay. In such a feeding assay, one exposesthe pest to a sample containing either compounds to be tested, orcontrol samples. Often this is performed by placing the material to betested, or a suitable dilution of such material, onto a material thatthe pest will ingest, such as an artificial diet. The material to betested may be composed of a liquid, solid, or slurry. The material to betested may be placed upon the surface and then allowed to dry.Alternatively, the material to be tested may be mixed with a moltenartificial diet, then dispensed into the assay chamber. The assaychamber may be, for example, a cup, a dish, or a well of a microtiterplate.

[0105] Assays for sucking pests (for example aphids) may involveseparating the test material from the insect by a partition, ideally aportion that can be pierced by the sucking mouth parts of the suckinginsect, to allow ingestion of the test material. Often the test materialis mixed with a feeding stimulant, such as sucrose, to promote ingestionof the test compound.

[0106] Other types of assays can include microinjection of the testmaterial into the mouth, or gut of the pest, as well as development oftransgenic plants, followed by test of the ability of the pest to feedupon the transgenic plant. Plant testing may involve isolation of theplant parts normally consumed, for example, small cages attached to aleaf, or isolation of entire plants in cages containing insects.

[0107] Other methods and approaches to assay pests are known in the art,and can be found, for example in Robertson, J. L. & H. K. Preisler.1992. Pesticide bioassays with arthropods. CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals “ArthropodManagement Tests” and “Journal of Economic Entomology” or by discussionwith members of the Entomological Society of America (ESA).

Example 9 Expression of AXMI-007 in Bacillus

[0108] The insecticidal AXMI-007 gene was amplified by PCR from pAX007,and cloned into the Bacillus expression vector pAX916 by methods wellknown in the art. The Bacillus strain containing pAX919 may be culturedon a variety of conventional growth media. A Bacillus strain containingpAX919 was grown in CYS media (10 g/l Bacto-casitone; 3 g/l yeastextract; 6 g/l KH₂PO₄; 14 g/l K₂HPO₄; 0.5 mM MgSO₄; 0.05 mM MnCl₂; 0.05mM FeSO₄), until sporulation was evident by microscopic examination.Samples were prepared, and AXMI-007 was tested for insecticidal activityin bioassays against important insect pests.

[0109] Methods

[0110] To prepare CYS media: 10 g/l Bacto-casitone; 3 g/l yeast extract;6 g/l KH₂PO₄; 14 g/l K₂HPO₄; 0.5 mM MgSO₄; 0.05 mM MnCl₂; 0.05 mM FeSO₄.The CYS mix should be pH 7, if adjustment is necessary. NaOH or HCl arepreferred. The media is then autoclaved and 100 ml of 10× filteredglucose is added after autoclaving. If the resultant solution is cloudyit can be stirred at room temperature to clear.

Example 10 Quantitation of AXMI-007 Insecticidal Activity Against Lyguslineolaris

[0111] Bacterial lysates were prepared by growing the Bacillus in 50 mlof CYS media for 60 hours. The Bacillus culture was then centrifuged at12,000 rpm for ten minutes and the supernatant discarded. The pellet wasresuspended in 5 ml of 20 mM Tris HCl at pH 8.

[0112] Bioassays were performed by cutting both the tip and the cap offan Eppendorf tube to form a feeding chamber. The insecticidal protein orcontrol was presented to the insect in a solution that was poured intothe cap and covered with parafilm (Pechiney Plastic Packaging, ChicagoIll.) that the insect could pierce upon feeding. The Eppendorf tube wasplaced back on the cap top down and 1^(st) or 2^(nd) instar Lygus nymphswere placed into the Eppendorf chamber with a fine tip brush. The cutEppendorf tube tip was sealed with parafilm creating an assay chamber.The resultant assay chamber was incubated at ambient temperature capside down. Insecticidal proteins were tested in a solution of 15%glucose at a concentration of 6.6 μg/ml. TABLE 2 Insecticidal Activityof AXMI-007 on Lygus lineolaris Protein No. Dead/Total % MortalityAXMI-007 3/6 50% Control 0/9 0%

Example 11 Vectoring of AXMI-007 for Plant Expression

[0113] The AXMI-007 coding region DNA is operably connected withappropriate promoter and terminator sequences for expression in plants.Such sequences are well known in the art and may include the rice actinpromoter or maize ubiquitin promoter for expression in monocots, theArabidopsis UBQ3 promoter or CaMV 35S promoter for expression in dicots,and the nos or PinII terminators. Techniques for producing andconfirming promoter—gene—terminator constructs also are well known inthe art.

[0114] The plant expression cassettes described above are combined withan appropriate plant selectable marker to aid in the selections oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation.

Example 12 Transformation of Maize Cells with AXMI-007

[0115] Maize ears are collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size are usedfor transformation. Embryos are plated scutellum side-up on a suitableincubation media, such as DN62A5S media (3.98 g/L N6 Salts; 1 mL/L (of1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine; 100 mg/L Myo-inositol;1.4 g/L L-Proline; 100 mg/L Casaminoacids; 50 g/L sucrose; 1 mL/L (of 1mg/mL Stock) 2,4-D), and incubated overnight at 25° C. in the dark.

[0116] The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

[0117] DNA constructs designed to express AXMI-007 in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for 30 min on osmotic media, thenplaced onto incubation media overnight at 25° C. in the dark. To avoidunduly damaging beamed explants, they are incubated for at least 24hours prior to transfer to recovery media. Embryos are then spread ontorecovery period media, for 5 days, 25° C. in the dark, then transferredto a selection media. Explants are incubated in selection media for upto eight weeks, depending on the nature and characteristics of theparticular selection utilized. After the selection period, the resultingcallus is transferred to embryo maturation media, until the formation ofmature somatic embryos is observed. The resulting mature somatic embryosare then placed under low light, and the process of regeneration isinitiated by methods known in the art. The resulting shoots are allowedto root on rooting media, and the resulting plants are transferred tonursery pots and propagated as transgenic plants.

[0118] Materials DN62A5S Media Components per liter Source Chu'S N6Basal 3.98 g/L Phytotechnology Labs Salt Mixture (Prod. No. C 416) Chu'sN6 1 mL/L (of 1000× Stock) Phytotechnology Labs Vitamin Solution (Prod.No. C 149) L-Asparagine 800 mg/L Phytotechnology Labs Myo-inositol 100mg/L Sigma L-Proline 1.4 g/L Phytotechnology Labs Casaminoacids 100 mg/LFisher Scientific Sucrose 50 g/L Phytotechnology Labs 2,4-D (Prod. No. 1mL/L (of 1 mg/mL Stock) Sigma D-7299)

[0119] Adjust the pH of the solution to pH to 5.8 with 1N KOH/1N KCl,add Gelrite (Sigma) to 3 g/L, and autoclave. After cooling to 50° C.,add 2 ml/L of a 5 mg/ml stock solution of Silver Nitrate(Phytotechnology Labs). Recipe yields about 20 plates.

Example 13 Transformation of AXMI-007 into Plant Cells byAgrobacterium-Mediated Transformation

[0120] Ears are collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size are usedfor transformation. Embryos are plated scutellum side-up on a suitableincubation media, and incubated overnight at 25° C. in the dark.However, it is not necessary per se to incubate the embryos overnight.Embryos are contacted with an Agrobacterium strain containing theappropriate vectors for Ti plasmid mediated transfer for 5-10 min, andthen plated onto co-cultivation media for 3 days (25° C. in the dark).After co-cultivation, explants are transferred to recovery period mediafor five days (at 25° C. in the dark). Explants are incubated inselection media for up to eight weeks, depending on the nature andcharacteristics of the particular selection utilized. After theselection period, the resulting callus is transferred to embryomaturation media, until the formation of mature somatic embryos isobserved. The resulting mature somatic embryos are then placed under lowlight, and the process of regeneration is initiated as known in the art.The resulting shoots are allowed to root on rooting media, and theresulting plants are transferred to nursery pots and propagated astransgenic plants.

[0121] All publications and patent applications mentioned in thespecification are indicative of the level of skill of those skilled inthe art to which this invention pertains. All publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application was specificallyand individually indicated to be incorporated by reference.

[0122] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1 17 1 2235 DNA Bacillus thuringiensis CDS (1)...(2235) 1 gtg aat caaaat aat aat aat gaa tat gag att atc gat tca aag aat 48 Met Asn Gln AsnAsn Asn Asn Glu Tyr Glu Ile Ile Asp Ser Lys Asn 1 5 10 15 tta tct tatcct tct aac aga aat att gat cat tct aga tac cct tac 96 Leu Ser Tyr ProSer Asn Arg Asn Ile Asp His Ser Arg Tyr Pro Tyr 20 25 30 aca aat aat ccaaat caa cca tta caa aac aca aat tac aaa gag tgg 144 Thr Asn Asn Pro AsnGln Pro Leu Gln Asn Thr Asn Tyr Lys Glu Trp 35 40 45 ctc aat atg tgt caaggg aat aca caa tat ggt gat aat ttc gag aca 192 Leu Asn Met Cys Gln GlyAsn Thr Gln Tyr Gly Asp Asn Phe Glu Thr 50 55 60 ttt gct agt gct gat acaatt gct gca gtt agt gca ggt act att gta 240 Phe Ala Ser Ala Asp Thr IleAla Ala Val Ser Ala Gly Thr Ile Val 65 70 75 80 tcc ggt act ctg tta gccggt ata ggt ggg ctc act tct ata tcc gga 288 Ser Gly Thr Leu Leu Ala GlyIle Gly Gly Leu Thr Ser Ile Ser Gly 85 90 95 ccg ata gga ata ata ggt gctata ata ata tct ttt ggt acc cta atc 336 Pro Ile Gly Ile Ile Gly Ala IleIle Ile Ser Phe Gly Thr Leu Ile 100 105 110 act gtc ttt tgg ccc gcg ggagaa caa gac aaa aca gta tgg aca caa 384 Thr Val Phe Trp Pro Ala Gly GluGln Asp Lys Thr Val Trp Thr Gln 115 120 125 ttt att aaa atg gga gaa attttt gtt gat aca ccg tta aca gaa agc 432 Phe Ile Lys Met Gly Glu Ile PheVal Asp Thr Pro Leu Thr Glu Ser 130 135 140 ata aaa cag cta aag tta caaact tta gaa gga ttt aga caa ata tta 480 Ile Lys Gln Leu Lys Leu Gln ThrLeu Glu Gly Phe Arg Gln Ile Leu 145 150 155 160 caa agc tat aat aca gcatta gat gat tgg aga aaa tta aaa aga cta 528 Gln Ser Tyr Asn Thr Ala LeuAsp Asp Trp Arg Lys Leu Lys Arg Leu 165 170 175 caa gct cct gga tta ccacca tca tca gca tta caa caa gct gcc ttg 576 Gln Ala Pro Gly Leu Pro ProSer Ser Ala Leu Gln Gln Ala Ala Leu 180 185 190 act ctt aaa ata cga tttgag aat gtt cac aat gat ttt att cga gaa 624 Thr Leu Lys Ile Arg Phe GluAsn Val His Asn Asp Phe Ile Arg Glu 195 200 205 ata cct ggt ttc caa cttgaa act tat aaa acg cta tta cta cct att 672 Ile Pro Gly Phe Gln Leu GluThr Tyr Lys Thr Leu Leu Leu Pro Ile 210 215 220 tat gcg caa gct gct aatttt cat tta aat tta tta caa caa ggt gct 720 Tyr Ala Gln Ala Ala Asn PheHis Leu Asn Leu Leu Gln Gln Gly Ala 225 230 235 240 gaa ttg gct gat gaatgg aat gca gat ata cat cct tca caa att gaa 768 Glu Leu Ala Asp Glu TrpAsn Ala Asp Ile His Pro Ser Gln Ile Glu 245 250 255 cct aat gct gga acatca gat gac tat tat aaa ctt tta aaa gaa aat 816 Pro Asn Ala Gly Thr SerAsp Asp Tyr Tyr Lys Leu Leu Lys Glu Asn 260 265 270 ata cct aaa tat agtaac tat tgt gca aat acc tat aga gaa gga cta 864 Ile Pro Lys Tyr Ser AsnTyr Cys Ala Asn Thr Tyr Arg Glu Gly Leu 275 280 285 aat aaa ctt cga aacgaa cct aat atg aga tgg agt ata ttt aat gat 912 Asn Lys Leu Arg Asn GluPro Asn Met Arg Trp Ser Ile Phe Asn Asp 290 295 300 tat cga aga tat atgact att act gta tta gat act atc gct caa ttt 960 Tyr Arg Arg Tyr Met ThrIle Thr Val Leu Asp Thr Ile Ala Gln Phe 305 310 315 320 tct ttt tat gatata aag aga tac aaa gat tca ata gga aga ata ggt 1008 Ser Phe Tyr Asp IleLys Arg Tyr Lys Asp Ser Ile Gly Arg Ile Gly 325 330 335 ggc att aaa actgaa ctt aca aga gaa att tat aca act gaa ata aat 1056 Gly Ile Lys Thr GluLeu Thr Arg Glu Ile Tyr Thr Thr Glu Ile Asn 340 345 350 ttt gac cgt cttact tac ctt gaa att caa ccc aat ctc gct ata atg 1104 Phe Asp Arg Leu ThrTyr Leu Glu Ile Gln Pro Asn Leu Ala Ile Met 355 360 365 gaa tat aat ttaaca cgt tca ggg ctt aga tta ttt tca ttt tta gat 1152 Glu Tyr Asn Leu ThrArg Ser Gly Leu Arg Leu Phe Ser Phe Leu Asp 370 375 380 gaa ctt ata ttttat aca aaa aat gaa acg tac ggg aat cgt tta gtt 1200 Glu Leu Ile Phe TyrThr Lys Asn Glu Thr Tyr Gly Asn Arg Leu Val 385 390 395 400 ggt att gcgaat cgt aat aga tct act tat gct acg aca gga act gaa 1248 Gly Ile Ala AsnArg Asn Arg Ser Thr Tyr Ala Thr Thr Gly Thr Glu 405 410 415 att ata tatgga gaa aga aca ggt cca ccc aca aca aaa act tta ata 1296 Ile Ile Tyr GlyGlu Arg Thr Gly Pro Pro Thr Thr Lys Thr Leu Ile 420 425 430 cca ttt gaatcc tat aaa gtt tca att gta act gat aga caa gta act 1344 Pro Phe Glu SerTyr Lys Val Ser Ile Val Thr Asp Arg Gln Val Thr 435 440 445 cct act tcccct ttt cct aac ata tac ttt aca att aat caa att gaa 1392 Pro Thr Ser ProPhe Pro Asn Ile Tyr Phe Thr Ile Asn Gln Ile Glu 450 455 460 ctt tat ttaaat aat tca cct agt aat aaa tta aca tat tca gct ggg 1440 Leu Tyr Leu AsnAsn Ser Pro Ser Asn Lys Leu Thr Tyr Ser Ala Gly 465 470 475 480 ggg aattta tct aat gat aaa aaa aca act gat ttt caa ttt cct gta 1488 Gly Asn LeuSer Asn Asp Lys Lys Thr Thr Asp Phe Gln Phe Pro Val 485 490 495 aaa aaagac tgt aaa cca att att aat cca aat tgt tta cca agc tat 1536 Lys Lys AspCys Lys Pro Ile Ile Asn Pro Asn Cys Leu Pro Ser Tyr 500 505 510 aat agttat agt cat att tta tcc cag ttt tct tta ttt aat tat tcc 1584 Asn Ser TyrSer His Ile Leu Ser Gln Phe Ser Leu Phe Asn Tyr Ser 515 520 525 tat aaaatt gga tta gcg cta aat ata tta tat aca ggt gca tta gga 1632 Tyr Lys IleGly Leu Ala Leu Asn Ile Leu Tyr Thr Gly Ala Leu Gly 530 535 540 tgg acacac agt agt gtt aat aga aat aat gca ata tca gat aaa ata 1680 Trp Thr HisSer Ser Val Asn Arg Asn Asn Ala Ile Ser Asp Lys Ile 545 550 555 560 attaca atg atc cca gca atc aaa ggt aac agt ctt gat aca aac tct 1728 Ile ThrMet Ile Pro Ala Ile Lys Gly Asn Ser Leu Asp Thr Asn Ser 565 570 575 aaggta att gaa gga cct ggt cat aca gga gga aac ttg gtt tat tta 1776 Lys ValIle Glu Gly Pro Gly His Thr Gly Gly Asn Leu Val Tyr Leu 580 585 590 caaagt caa ggg cgt tta gag att aca tgt aga act cct aat tct aca 1824 Gln SerGln Gly Arg Leu Glu Ile Thr Cys Arg Thr Pro Asn Ser Thr 595 600 605 caatct tat tac att aga ctt cga tac gct aca aat ggt gct gga aat 1872 Gln SerTyr Tyr Ile Arg Leu Arg Tyr Ala Thr Asn Gly Ala Gly Asn 610 615 620 actctt cct aat ata tct ctt aca ata cca gga gta ata gga ata cca 1920 Thr LeuPro Asn Ile Ser Leu Thr Ile Pro Gly Val Ile Gly Ile Pro 625 630 635 640cct caa cga ctc aac aac act ttt tct ggt aca aat tat aat aat tta 1968 ProGln Arg Leu Asn Asn Thr Phe Ser Gly Thr Asn Tyr Asn Asn Leu 645 650 655caa tac gga gat ttt ggg tat ttc caa ttt cca agt aca gta aca tta 2016 GlnTyr Gly Asp Phe Gly Tyr Phe Gln Phe Pro Ser Thr Val Thr Leu 660 665 670cct tta aat cga aac ata cca ttt ata ttt aat cgt gca gat gta tca 2064 ProLeu Asn Arg Asn Ile Pro Phe Ile Phe Asn Arg Ala Asp Val Ser 675 680 685aat tca att tta atc att gat aaa att gaa ttt ata cca att act tcc 2112 AsnSer Ile Leu Ile Ile Asp Lys Ile Glu Phe Ile Pro Ile Thr Ser 690 695 700tct gta cgc caa aat aga gaa aaa caa aaa tta gaa act atc caa aca 2160 SerVal Arg Gln Asn Arg Glu Lys Gln Lys Leu Glu Thr Ile Gln Thr 705 710 715720 aaa ata aat aca ttt ttc aca aat cat aca aaa aat act tta aat ata 2208Lys Ile Asn Thr Phe Phe Thr Asn His Thr Lys Asn Thr Leu Asn Ile 725 730735 gaa gcc aca aac tat gat att gat taa 2235 Glu Ala Thr Asn Tyr Asp IleAsp * 740 2 744 PRT Bacillus thuringiensis 2 Met Asn Gln Asn Asn Asn AsnGlu Tyr Glu Ile Ile Asp Ser Lys Asn 1 5 10 15 Leu Ser Tyr Pro Ser AsnArg Asn Ile Asp His Ser Arg Tyr Pro Tyr 20 25 30 Thr Asn Asn Pro Asn GlnPro Leu Gln Asn Thr Asn Tyr Lys Glu Trp 35 40 45 Leu Asn Met Cys Gln GlyAsn Thr Gln Tyr Gly Asp Asn Phe Glu Thr 50 55 60 Phe Ala Ser Ala Asp ThrIle Ala Ala Val Ser Ala Gly Thr Ile Val 65 70 75 80 Ser Gly Thr Leu LeuAla Gly Ile Gly Gly Leu Thr Ser Ile Ser Gly 85 90 95 Pro Ile Gly Ile IleGly Ala Ile Ile Ile Ser Phe Gly Thr Leu Ile 100 105 110 Thr Val Phe TrpPro Ala Gly Glu Gln Asp Lys Thr Val Trp Thr Gln 115 120 125 Phe Ile LysMet Gly Glu Ile Phe Val Asp Thr Pro Leu Thr Glu Ser 130 135 140 Ile LysGln Leu Lys Leu Gln Thr Leu Glu Gly Phe Arg Gln Ile Leu 145 150 155 160Gln Ser Tyr Asn Thr Ala Leu Asp Asp Trp Arg Lys Leu Lys Arg Leu 165 170175 Gln Ala Pro Gly Leu Pro Pro Ser Ser Ala Leu Gln Gln Ala Ala Leu 180185 190 Thr Leu Lys Ile Arg Phe Glu Asn Val His Asn Asp Phe Ile Arg Glu195 200 205 Ile Pro Gly Phe Gln Leu Glu Thr Tyr Lys Thr Leu Leu Leu ProIle 210 215 220 Tyr Ala Gln Ala Ala Asn Phe His Leu Asn Leu Leu Gln GlnGly Ala 225 230 235 240 Glu Leu Ala Asp Glu Trp Asn Ala Asp Ile His ProSer Gln Ile Glu 245 250 255 Pro Asn Ala Gly Thr Ser Asp Asp Tyr Tyr LysLeu Leu Lys Glu Asn 260 265 270 Ile Pro Lys Tyr Ser Asn Tyr Cys Ala AsnThr Tyr Arg Glu Gly Leu 275 280 285 Asn Lys Leu Arg Asn Glu Pro Asn MetArg Trp Ser Ile Phe Asn Asp 290 295 300 Tyr Arg Arg Tyr Met Thr Ile ThrVal Leu Asp Thr Ile Ala Gln Phe 305 310 315 320 Ser Phe Tyr Asp Ile LysArg Tyr Lys Asp Ser Ile Gly Arg Ile Gly 325 330 335 Gly Ile Lys Thr GluLeu Thr Arg Glu Ile Tyr Thr Thr Glu Ile Asn 340 345 350 Phe Asp Arg LeuThr Tyr Leu Glu Ile Gln Pro Asn Leu Ala Ile Met 355 360 365 Glu Tyr AsnLeu Thr Arg Ser Gly Leu Arg Leu Phe Ser Phe Leu Asp 370 375 380 Glu LeuIle Phe Tyr Thr Lys Asn Glu Thr Tyr Gly Asn Arg Leu Val 385 390 395 400Gly Ile Ala Asn Arg Asn Arg Ser Thr Tyr Ala Thr Thr Gly Thr Glu 405 410415 Ile Ile Tyr Gly Glu Arg Thr Gly Pro Pro Thr Thr Lys Thr Leu Ile 420425 430 Pro Phe Glu Ser Tyr Lys Val Ser Ile Val Thr Asp Arg Gln Val Thr435 440 445 Pro Thr Ser Pro Phe Pro Asn Ile Tyr Phe Thr Ile Asn Gln IleGlu 450 455 460 Leu Tyr Leu Asn Asn Ser Pro Ser Asn Lys Leu Thr Tyr SerAla Gly 465 470 475 480 Gly Asn Leu Ser Asn Asp Lys Lys Thr Thr Asp PheGln Phe Pro Val 485 490 495 Lys Lys Asp Cys Lys Pro Ile Ile Asn Pro AsnCys Leu Pro Ser Tyr 500 505 510 Asn Ser Tyr Ser His Ile Leu Ser Gln PheSer Leu Phe Asn Tyr Ser 515 520 525 Tyr Lys Ile Gly Leu Ala Leu Asn IleLeu Tyr Thr Gly Ala Leu Gly 530 535 540 Trp Thr His Ser Ser Val Asn ArgAsn Asn Ala Ile Ser Asp Lys Ile 545 550 555 560 Ile Thr Met Ile Pro AlaIle Lys Gly Asn Ser Leu Asp Thr Asn Ser 565 570 575 Lys Val Ile Glu GlyPro Gly His Thr Gly Gly Asn Leu Val Tyr Leu 580 585 590 Gln Ser Gln GlyArg Leu Glu Ile Thr Cys Arg Thr Pro Asn Ser Thr 595 600 605 Gln Ser TyrTyr Ile Arg Leu Arg Tyr Ala Thr Asn Gly Ala Gly Asn 610 615 620 Thr LeuPro Asn Ile Ser Leu Thr Ile Pro Gly Val Ile Gly Ile Pro 625 630 635 640Pro Gln Arg Leu Asn Asn Thr Phe Ser Gly Thr Asn Tyr Asn Asn Leu 645 650655 Gln Tyr Gly Asp Phe Gly Tyr Phe Gln Phe Pro Ser Thr Val Thr Leu 660665 670 Pro Leu Asn Arg Asn Ile Pro Phe Ile Phe Asn Arg Ala Asp Val Ser675 680 685 Asn Ser Ile Leu Ile Ile Asp Lys Ile Glu Phe Ile Pro Ile ThrSer 690 695 700 Ser Val Arg Gln Asn Arg Glu Lys Gln Lys Leu Glu Thr IleGln Thr 705 710 715 720 Lys Ile Asn Thr Phe Phe Thr Asn His Thr Lys AsnThr Leu Asn Ile 725 730 735 Glu Ala Thr Asn Tyr Asp Ile Asp 740 3 2085DNA Bacillus thuringiensis CDS (1)...(2085) 3 atg tgt caa ggg aat acacaa tat ggt gat aat ttc gag aca ttt gct 48 Met Cys Gln Gly Asn Thr GlnTyr Gly Asp Asn Phe Glu Thr Phe Ala 1 5 10 15 agt gct gat aca att gctgca gtt agt gca ggt act att gta tcc ggt 96 Ser Ala Asp Thr Ile Ala AlaVal Ser Ala Gly Thr Ile Val Ser Gly 20 25 30 act ctg tta gcc ggt ata ggtggg ctc act tct ata tcc gga ccg ata 144 Thr Leu Leu Ala Gly Ile Gly GlyLeu Thr Ser Ile Ser Gly Pro Ile 35 40 45 gga ata ata ggt gct ata ata atatct ttt ggt acc cta atc act gtc 192 Gly Ile Ile Gly Ala Ile Ile Ile SerPhe Gly Thr Leu Ile Thr Val 50 55 60 ttt tgg ccc gcg gga gaa caa gac aaaaca gta tgg aca caa ttt att 240 Phe Trp Pro Ala Gly Glu Gln Asp Lys ThrVal Trp Thr Gln Phe Ile 65 70 75 80 aaa atg gga gaa att ttt gtt gat acaccg tta aca gaa agc ata aaa 288 Lys Met Gly Glu Ile Phe Val Asp Thr ProLeu Thr Glu Ser Ile Lys 85 90 95 cag cta aag tta caa act tta gaa gga tttaga caa ata tta caa agc 336 Gln Leu Lys Leu Gln Thr Leu Glu Gly Phe ArgGln Ile Leu Gln Ser 100 105 110 tat aat aca gca tta gat gat tgg aga aaatta aaa aga cta caa gct 384 Tyr Asn Thr Ala Leu Asp Asp Trp Arg Lys LeuLys Arg Leu Gln Ala 115 120 125 cct gga tta cca cca tca tca gca tta caacaa gct gcc ttg act ctt 432 Pro Gly Leu Pro Pro Ser Ser Ala Leu Gln GlnAla Ala Leu Thr Leu 130 135 140 aaa ata cga ttt gag aat gtt cac aat gatttt att cga gaa ata cct 480 Lys Ile Arg Phe Glu Asn Val His Asn Asp PheIle Arg Glu Ile Pro 145 150 155 160 ggt ttc caa ctt gaa act tat aaa acgcta tta cta cct att tat gcg 528 Gly Phe Gln Leu Glu Thr Tyr Lys Thr LeuLeu Leu Pro Ile Tyr Ala 165 170 175 caa gct gct aat ttt cat tta aat ttatta caa caa ggt gct gaa ttg 576 Gln Ala Ala Asn Phe His Leu Asn Leu LeuGln Gln Gly Ala Glu Leu 180 185 190 gct gat gaa tgg aat gca gat ata catcct tca caa att gaa cct aat 624 Ala Asp Glu Trp Asn Ala Asp Ile His ProSer Gln Ile Glu Pro Asn 195 200 205 gct gga aca tca gat gac tat tat aaactt tta aaa gaa aat ata cct 672 Ala Gly Thr Ser Asp Asp Tyr Tyr Lys LeuLeu Lys Glu Asn Ile Pro 210 215 220 aaa tat agt aac tat tgt gca aat acctat aga gaa gga cta aat aaa 720 Lys Tyr Ser Asn Tyr Cys Ala Asn Thr TyrArg Glu Gly Leu Asn Lys 225 230 235 240 ctt cga aac gaa cct aat atg agatgg agt ata ttt aat gat tat cga 768 Leu Arg Asn Glu Pro Asn Met Arg TrpSer Ile Phe Asn Asp Tyr Arg 245 250 255 aga tat atg act att act gta ttagat act atc gct caa ttt tct ttt 816 Arg Tyr Met Thr Ile Thr Val Leu AspThr Ile Ala Gln Phe Ser Phe 260 265 270 tat gat ata aag aga tac aaa gattca ata gga aga ata ggt ggc att 864 Tyr Asp Ile Lys Arg Tyr Lys Asp SerIle Gly Arg Ile Gly Gly Ile 275 280 285 aaa act gaa ctt aca aga gaa atttat aca act gaa ata aat ttt gac 912 Lys Thr Glu Leu Thr Arg Glu Ile TyrThr Thr Glu Ile Asn Phe Asp 290 295 300 cgt ctt act tac ctt gaa att caaccc aat ctc gct ata atg gaa tat 960 Arg Leu Thr Tyr Leu Glu Ile Gln ProAsn Leu Ala Ile Met Glu Tyr 305 310 315 320 aat tta aca cgt tca ggg cttaga tta ttt tca ttt tta gat gaa ctt 1008 Asn Leu Thr Arg Ser Gly Leu ArgLeu Phe Ser Phe Leu Asp Glu Leu 325 330 335 ata ttt tat aca aaa aat gaaacg tac ggg aat cgt tta gtt ggt att 1056 Ile Phe Tyr Thr Lys Asn Glu ThrTyr Gly Asn Arg Leu Val Gly Ile 340 345 350 gcg aat cgt aat aga tct acttat gct acg aca gga act gaa att ata 1104 Ala Asn Arg Asn Arg Ser Thr TyrAla Thr Thr Gly Thr Glu Ile Ile 355 360 365 tat gga gaa aga aca ggt ccaccc aca aca aaa act tta ata cca ttt 1152 Tyr Gly Glu Arg Thr Gly Pro ProThr Thr Lys Thr Leu Ile Pro Phe 370 375 380 gaa tcc tat aaa gtt tca attgta act gat aga caa gta act cct act 1200 Glu Ser Tyr Lys Val Ser Ile ValThr Asp Arg Gln Val Thr Pro Thr 385 390 395 400 tcc cct ttt cct aac atatac ttt aca att aat caa att gaa ctt tat 1248 Ser Pro Phe Pro Asn Ile TyrPhe Thr Ile Asn Gln Ile Glu Leu Tyr 405 410 415 tta aat aat tca cct agtaat aaa tta aca tat tca gct ggg ggg aat 1296 Leu Asn Asn Ser Pro Ser AsnLys Leu Thr Tyr Ser Ala Gly Gly Asn 420 425 430 tta tct aat gat aaa aaaaca act gat ttt caa ttt cct gta aaa aaa 1344 Leu Ser Asn Asp Lys Lys ThrThr Asp Phe Gln Phe Pro Val Lys Lys 435 440 445 gac tgt aaa cca att attaat cca aat tgt tta cca agc tat aat agt 1392 Asp Cys Lys Pro Ile Ile AsnPro Asn Cys Leu Pro Ser Tyr Asn Ser 450 455 460 tat agt cat att tta tcccag ttt tct tta ttt aat tat tcc tat aaa 1440 Tyr Ser His Ile Leu Ser GlnPhe Ser Leu Phe Asn Tyr Ser Tyr Lys 465 470 475 480 att gga tta gcg ctaaat ata tta tat aca ggt gca tta gga tgg aca 1488 Ile Gly Leu Ala Leu AsnIle Leu Tyr Thr Gly Ala Leu Gly Trp Thr 485 490 495 cac agt agt gtt aataga aat aat gca ata tca gat aaa ata att aca 1536 His Ser Ser Val Asn ArgAsn Asn Ala Ile Ser Asp Lys Ile Ile Thr 500 505 510 atg atc cca gca atcaaa ggt aac agt ctt gat aca aac tct aag gta 1584 Met Ile Pro Ala Ile LysGly Asn Ser Leu Asp Thr Asn Ser Lys Val 515 520 525 att gaa gga cct ggtcat aca gga gga aac ttg gtt tat tta caa agt 1632 Ile Glu Gly Pro Gly HisThr Gly Gly Asn Leu Val Tyr Leu Gln Ser 530 535 540 caa ggg cgt tta gagatt aca tgt aga act cct aat tct aca caa tct 1680 Gln Gly Arg Leu Glu IleThr Cys Arg Thr Pro Asn Ser Thr Gln Ser 545 550 555 560 tat tac att agactt cga tac gct aca aat ggt gct gga aat act ctt 1728 Tyr Tyr Ile Arg LeuArg Tyr Ala Thr Asn Gly Ala Gly Asn Thr Leu 565 570 575 cct aat ata tctctt aca ata cca gga gta ata gga ata cca cct caa 1776 Pro Asn Ile Ser LeuThr Ile Pro Gly Val Ile Gly Ile Pro Pro Gln 580 585 590 cga ctc aac aacact ttt tct ggt aca aat tat aat aat tta caa tac 1824 Arg Leu Asn Asn ThrPhe Ser Gly Thr Asn Tyr Asn Asn Leu Gln Tyr 595 600 605 gga gat ttt gggtat ttc caa ttt cca agt aca gta aca tta cct tta 1872 Gly Asp Phe Gly TyrPhe Gln Phe Pro Ser Thr Val Thr Leu Pro Leu 610 615 620 aat cga aac atacca ttt ata ttt aat cgt gca gat gta tca aat tca 1920 Asn Arg Asn Ile ProPhe Ile Phe Asn Arg Ala Asp Val Ser Asn Ser 625 630 635 640 att tta atcatt gat aaa att gaa ttt ata cca att act tcc tct gta 1968 Ile Leu Ile IleAsp Lys Ile Glu Phe Ile Pro Ile Thr Ser Ser Val 645 650 655 cgc caa aataga gaa aaa caa aaa tta gaa act atc caa aca aaa ata 2016 Arg Gln Asn ArgGlu Lys Gln Lys Leu Glu Thr Ile Gln Thr Lys Ile 660 665 670 aat aca tttttc aca aat cat aca aaa aat act tta aat ata gaa gcc 2064 Asn Thr Phe PheThr Asn His Thr Lys Asn Thr Leu Asn Ile Glu Ala 675 680 685 aca aac tatgat att gat taa 2085 Thr Asn Tyr Asp Ile Asp * 690 4 694 PRT Bacillusthuringiensis 4 Met Cys Gln Gly Asn Thr Gln Tyr Gly Asp Asn Phe Glu ThrPhe Ala 1 5 10 15 Ser Ala Asp Thr Ile Ala Ala Val Ser Ala Gly Thr IleVal Ser Gly 20 25 30 Thr Leu Leu Ala Gly Ile Gly Gly Leu Thr Ser Ile SerGly Pro Ile 35 40 45 Gly Ile Ile Gly Ala Ile Ile Ile Ser Phe Gly Thr LeuIle Thr Val 50 55 60 Phe Trp Pro Ala Gly Glu Gln Asp Lys Thr Val Trp ThrGln Phe Ile 65 70 75 80 Lys Met Gly Glu Ile Phe Val Asp Thr Pro Leu ThrGlu Ser Ile Lys 85 90 95 Gln Leu Lys Leu Gln Thr Leu Glu Gly Phe Arg GlnIle Leu Gln Ser 100 105 110 Tyr Asn Thr Ala Leu Asp Asp Trp Arg Lys LeuLys Arg Leu Gln Ala 115 120 125 Pro Gly Leu Pro Pro Ser Ser Ala Leu GlnGln Ala Ala Leu Thr Leu 130 135 140 Lys Ile Arg Phe Glu Asn Val His AsnAsp Phe Ile Arg Glu Ile Pro 145 150 155 160 Gly Phe Gln Leu Glu Thr TyrLys Thr Leu Leu Leu Pro Ile Tyr Ala 165 170 175 Gln Ala Ala Asn Phe HisLeu Asn Leu Leu Gln Gln Gly Ala Glu Leu 180 185 190 Ala Asp Glu Trp AsnAla Asp Ile His Pro Ser Gln Ile Glu Pro Asn 195 200 205 Ala Gly Thr SerAsp Asp Tyr Tyr Lys Leu Leu Lys Glu Asn Ile Pro 210 215 220 Lys Tyr SerAsn Tyr Cys Ala Asn Thr Tyr Arg Glu Gly Leu Asn Lys 225 230 235 240 LeuArg Asn Glu Pro Asn Met Arg Trp Ser Ile Phe Asn Asp Tyr Arg 245 250 255Arg Tyr Met Thr Ile Thr Val Leu Asp Thr Ile Ala Gln Phe Ser Phe 260 265270 Tyr Asp Ile Lys Arg Tyr Lys Asp Ser Ile Gly Arg Ile Gly Gly Ile 275280 285 Lys Thr Glu Leu Thr Arg Glu Ile Tyr Thr Thr Glu Ile Asn Phe Asp290 295 300 Arg Leu Thr Tyr Leu Glu Ile Gln Pro Asn Leu Ala Ile Met GluTyr 305 310 315 320 Asn Leu Thr Arg Ser Gly Leu Arg Leu Phe Ser Phe LeuAsp Glu Leu 325 330 335 Ile Phe Tyr Thr Lys Asn Glu Thr Tyr Gly Asn ArgLeu Val Gly Ile 340 345 350 Ala Asn Arg Asn Arg Ser Thr Tyr Ala Thr ThrGly Thr Glu Ile Ile 355 360 365 Tyr Gly Glu Arg Thr Gly Pro Pro Thr ThrLys Thr Leu Ile Pro Phe 370 375 380 Glu Ser Tyr Lys Val Ser Ile Val ThrAsp Arg Gln Val Thr Pro Thr 385 390 395 400 Ser Pro Phe Pro Asn Ile TyrPhe Thr Ile Asn Gln Ile Glu Leu Tyr 405 410 415 Leu Asn Asn Ser Pro SerAsn Lys Leu Thr Tyr Ser Ala Gly Gly Asn 420 425 430 Leu Ser Asn Asp LysLys Thr Thr Asp Phe Gln Phe Pro Val Lys Lys 435 440 445 Asp Cys Lys ProIle Ile Asn Pro Asn Cys Leu Pro Ser Tyr Asn Ser 450 455 460 Tyr Ser HisIle Leu Ser Gln Phe Ser Leu Phe Asn Tyr Ser Tyr Lys 465 470 475 480 IleGly Leu Ala Leu Asn Ile Leu Tyr Thr Gly Ala Leu Gly Trp Thr 485 490 495His Ser Ser Val Asn Arg Asn Asn Ala Ile Ser Asp Lys Ile Ile Thr 500 505510 Met Ile Pro Ala Ile Lys Gly Asn Ser Leu Asp Thr Asn Ser Lys Val 515520 525 Ile Glu Gly Pro Gly His Thr Gly Gly Asn Leu Val Tyr Leu Gln Ser530 535 540 Gln Gly Arg Leu Glu Ile Thr Cys Arg Thr Pro Asn Ser Thr GlnSer 545 550 555 560 Tyr Tyr Ile Arg Leu Arg Tyr Ala Thr Asn Gly Ala GlyAsn Thr Leu 565 570 575 Pro Asn Ile Ser Leu Thr Ile Pro Gly Val Ile GlyIle Pro Pro Gln 580 585 590 Arg Leu Asn Asn Thr Phe Ser Gly Thr Asn TyrAsn Asn Leu Gln Tyr 595 600 605 Gly Asp Phe Gly Tyr Phe Gln Phe Pro SerThr Val Thr Leu Pro Leu 610 615 620 Asn Arg Asn Ile Pro Phe Ile Phe AsnArg Ala Asp Val Ser Asn Ser 625 630 635 640 Ile Leu Ile Ile Asp Lys IleGlu Phe Ile Pro Ile Thr Ser Ser Val 645 650 655 Arg Gln Asn Arg Glu LysGln Lys Leu Glu Thr Ile Gln Thr Lys Ile 660 665 670 Asn Thr Phe Phe ThrAsn His Thr Lys Asn Thr Leu Asn Ile Glu Ala 675 680 685 Thr Asn Tyr AspIle Asp 690 5 1176 PRT Bacillus thuringiensis 5 Met Asp Asn Asn Pro AsnIle Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 10 15 Ser Asn Pro Glu ValGlu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 25 30 Tyr Thr Pro Ile AspIle Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 40 45 Glu Phe Val Pro GlyAla Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 55 60 Trp Gly Ile Phe GlyPro Ser Gln Trp Asp Ala Phe Pro Val Gln Ile 65 70 75 80 Glu Gln Leu IleAsn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 85 90 95 Ile Ser Arg LeuGlu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu 100 105 110 Ser Phe ArgGlu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu Arg Glu 115 120 125 Glu MetArg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu Thr Thr Ala 130 135 140 IlePro Leu Leu Ala Val Gln Asn Tyr Gln Val Pro Leu Leu Ser Val 145 150 155160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser Val Leu Arg Asp Val Ser 165170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp Ala Ala Thr Ile Asn Ser Arg180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile Gly Asn Tyr Thr Asp Tyr AlaVal 195 200 205 Arg Trp Tyr Asn Thr Gly Leu Glu Arg Val Trp Gly Pro AspSer Arg 210 215 220 Asp Trp Val Arg Tyr Asn Gln Phe Arg Arg Glu Leu ThrLeu Thr Val 225 230 235 240 Leu Asp Ile Val Ala Leu Phe Ser Asn Tyr AspSer Arg Arg Tyr Pro 245 250 255 Ile Arg Thr Val Ser Gln Leu Thr Arg GluIle Tyr Thr Asn Pro Val 260 265 270 Leu Glu Asn Phe Asp Gly Ser Phe ArgGly Met Ala Gln Arg Ile Glu 275 280 285 Gln Asn Ile Arg Gln Pro His LeuMet Asp Ile Leu Asn Ser Ile Thr 290 295 300 Ile Tyr Thr Asp Val His ArgGly Phe Asn Tyr Trp Ser Gly His Gln 305 310 315 320 Ile Thr Ala Ser ProVal Gly Phe Ser Gly Pro Glu Phe Ala Phe Pro 325 330 335 Leu Phe Gly AsnAla Gly Asn Ala Ala Pro Pro Val Leu Val Ser Leu 340 345 350 Thr Gly LeuGly Ile Phe Arg Thr Leu Ser Ser Pro Leu Tyr Arg Arg 355 360 365 Ile IleLeu Gly Ser Gly Pro Asn Asn Gln Glu Leu Phe Val Leu Asp 370 375 380 GlyThr Glu Phe Ser Phe Ala Ser Leu Thr Thr Asn Leu Pro Ser Thr 385 390 395400 Ile Tyr Arg Gln Arg Gly Thr Val Asp Ser Leu Asp Val Ile Pro Pro 405410 415 Gln Asp Asn Ser Val Pro Pro Arg Ala Gly Phe Ser His Arg Leu Ser420 425 430 His Val Thr Met Leu Ser Gln Ala Ala Gly Ala Val Tyr Thr LeuArg 435 440 445 Ala Pro Thr Phe Ser Trp Gln His Arg Ser Ala Glu Phe AsnAsn Ile 450 455 460 Ile Pro Ser Ser Gln Ile Thr Gln Ile Pro Leu Thr LysSer Thr Asn 465 470 475 480 Leu Gly Ser Gly Thr Ser Val Val Lys Gly ProGly Phe Thr Gly Gly 485 490 495 Asp Ile Leu Arg Arg Thr Ser Pro Gly GlnIle Ser Thr Leu Arg Val 500 505 510 Asn Ile Thr Ala Pro Leu Ser Gln ArgTyr Arg Val Arg Ile Arg Tyr 515 520 525 Ala Ser Thr Thr Asn Leu Gln PheHis Thr Ser Ile Asp Gly Arg Pro 530 535 540 Ile Asn Gln Gly Asn Phe SerAla Thr Met Ser Ser Gly Ser Asn Leu 545 550 555 560 Gln Ser Gly Ser PheArg Thr Val Gly Phe Thr Thr Pro Phe Asn Phe 565 570 575 Ser Asn Gly SerSer Val Phe Thr Leu Ser Ala His Val Phe Asn Ser 580 585 590 Gly Asn GluVal Tyr Ile Asp Arg Ile Glu Phe Val Pro Ala Glu Val 595 600 605 Thr PheGlu Ala Glu Tyr Asp Leu Glu Arg Ala Gln Lys Ala Val Asn 610 615 620 GluLeu Phe Thr Ser Ser Asn Gln Ile Gly Leu Lys Thr Asp Val Thr 625 630 635640 Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val Glu Cys Leu Ser Asp 645650 655 Glu Phe Cys Leu Asp Glu Lys Gln Glu Leu Ser Glu Lys Val Lys His660 665 670 Ala Lys Arg Leu Ser Asp Glu Arg Asn Leu Leu Gln Asp Pro AsnPhe 675 680 685 Arg Gly Ile Asn Arg Gln Leu Asp Arg Gly Trp Arg Gly SerThr Asp 690 695 700 Ile Thr Ile Gln Gly Gly Asp Asp Val Phe Lys Glu AsnTyr Val Thr 705 710 715 720 Leu Leu Gly Thr Phe Asp Glu Cys Tyr Pro ThrTyr Leu Tyr Gln Lys 725 730 735 Ile Asp Glu Ser Lys Leu Lys Ala Tyr ThrArg Tyr Gln Leu Arg Gly 740 745 750 Tyr Ile Glu Asp Ser Gln Asp Leu GluIle Tyr Leu Ile Arg Tyr Asn 755 760 765 Ala Lys His Glu Thr Val Asn ValPro Gly Thr Gly Ser Leu Trp Pro 770 775 780 Leu Ser Ala Gln Ser Pro IleGly Lys Cys Gly Glu Pro Asn Arg Cys 785 790 795 800 Ala Pro His Leu GluTrp Asn Pro Asp Leu Asp Cys Ser Cys Arg Asp 805 810 815 Gly Glu Lys CysAla His His Ser His His Phe Ser Leu Asp Ile Asp 820 825 830 Val Gly CysThr Asp Leu Asn Glu Asp Leu Gly Val Trp Val Ile Phe 835 840 845 Lys IleLys Thr Gln Asp Gly His Ala Arg Leu Gly Asn Leu Glu Phe 850 855 860 LeuGlu Glu Lys Pro Leu Val Gly Glu Ala Leu Ala Arg Val Lys Arg 865 870 875880 Ala Glu Lys Lys Trp Arg Asp Lys Arg Glu Lys Leu Glu Trp Glu Thr 885890 895 Asn Ile Val Tyr Lys Glu Ala Lys Glu Ser Val Asp Ala Leu Phe Val900 905 910 Asn Ser Gln Tyr Asp Gln Leu Gln Ala Asp Thr Asn Ile Ala MetIle 915 920 925 His Ala Ala Asp Lys Arg Val His Ser Ile Arg Glu Ala TyrLeu Pro 930 935 940 Glu Leu Ser Val Ile Pro Gly Val Asn Ala Ala Ile PheGlu Glu Leu 945 950 955 960 Glu Gly Arg Ile Phe Thr Ala Phe Ser Leu TyrAsp Ala Arg Asn Val 965 970 975 Ile Lys Asn Gly Asp Phe Asn Asn Gly LeuSer Cys Trp Asn Val Lys 980 985 990 Gly His Val Asp Val Glu Glu Gln AsnAsn Gln Arg Ser Val Leu Val 995 1000 1005 Val Pro Glu Trp Glu Ala GluVal Ser Gln Glu Val Arg Val Cys Pro 1010 1015 1020 Gly Arg Gly Tyr IleLeu Arg Val Thr Ala Tyr Lys Glu Gly Tyr Gly 1025 1030 1035 1040 Glu GlyCys Val Thr Ile His Glu Ile Glu Asn Asn Thr Asp Glu Leu 1045 1050 1055Lys Phe Ser Asn Cys Val Glu Glu Glu Ile Tyr Pro Asn Asn Thr Val 10601065 1070 Thr Cys Asn Asp Tyr Thr Val Asn Gln Glu Glu Tyr Gly Gly AlaTyr 1075 1080 1085 Thr Ser Arg Asn Arg Gly Tyr Asn Glu Ala Pro Ser ValPro Ala Asp 1090 1095 1100 Tyr Ala Ser Val Tyr Glu Glu Lys Ser Tyr ThrAsp Gly Arg Arg Glu 1105 1110 1115 1120 Asn Pro Cys Glu Phe Asn Arg GlyTyr Arg Asp Tyr Thr Pro Leu Pro 1125 1130 1135 Val Gly Tyr Val Thr LysGlu Leu Glu Tyr Phe Pro Glu Thr Asp Lys 1140 1145 1150 Val Trp Ile GluIle Gly Glu Thr Glu Gly Thr Phe Ile Val Asp Ser 1155 1160 1165 Val GluLeu Leu Leu Met Glu Glu 1170 1175 6 1178 PRT Bacillus thuringiensis 6Met Asp Asn Asn Pro Asn Ile Asn Glu Cys Ile Pro Tyr Asn Cys Leu 1 5 1015 Ser Asn Pro Glu Val Glu Val Leu Gly Gly Glu Arg Ile Glu Thr Gly 20 2530 Tyr Thr Pro Ile Asp Ile Ser Leu Ser Leu Thr Gln Phe Leu Leu Ser 35 4045 Glu Phe Val Pro Gly Ala Gly Phe Val Leu Gly Leu Val Asp Ile Ile 50 5560 Trp Gly Ile Phe Gly Pro Ser Gln Trp Asp Ala Phe Leu Val Gln Ile 65 7075 80 Glu Gln Leu Ile Asn Gln Arg Ile Glu Glu Phe Ala Arg Asn Gln Ala 8590 95 Ile Ser Arg Leu Glu Gly Leu Ser Asn Leu Tyr Gln Ile Tyr Ala Glu100 105 110 Ser Phe Arg Glu Trp Glu Ala Asp Pro Thr Asn Pro Ala Leu ArgGlu 115 120 125 Glu Met Arg Ile Gln Phe Asn Asp Met Asn Ser Ala Leu ThrThr Ala 130 135 140 Ile Pro Leu Phe Ala Val Gln Asn Tyr Gln Val Pro LeuLeu Ser Val 145 150 155 160 Tyr Val Gln Ala Ala Asn Leu His Leu Ser ValLeu Arg Asp Val Ser 165 170 175 Val Phe Gly Gln Arg Trp Gly Phe Asp AlaAla Thr Ile Asn Ser Arg 180 185 190 Tyr Asn Asp Leu Thr Arg Leu Ile GlyAsn Tyr Thr Asp Tyr Ala Val 195 200 205 Arg Trp Tyr Asn Thr Gly Leu GluArg Val Trp Gly Pro Asp Ser Arg 210 215 220 Asp Trp Val Arg Tyr Asn GlnPhe Arg Arg Glu Leu Thr Leu Thr Val 225 230 235 240 Leu Asp Ile Val AlaLeu Phe Pro Asn Tyr Asp Ser Arg Arg Tyr Pro 245 250 255 Ile Arg Thr ValSer Gln Leu Thr Arg Glu Ile Tyr Thr Asn Pro Val 260 265 270 Leu Glu AsnPhe Asp Gly Ser Phe Arg Gly Ser Ala Gln Gly Ile Glu 275 280 285 Arg SerIle Arg Ser Pro His Leu Met Asp Ile Leu Asn Ser Ile Thr 290 295 300 IleTyr Thr Asp Ala His Arg Gly Tyr Tyr Tyr Trp Ser Gly His Gln 305 310 315320 Ile Met Ala Ser Pro Val Gly Phe Ser Gly Pro Glu Phe Thr Phe Pro 325330 335 Leu Tyr Gly Thr Met Gly Asn Ala Ala Pro Gln Gln Arg Ile Val Ala340 345 350 Gln Leu Gly Gln Gly Val Tyr Arg Thr Leu Ser Ser Thr Leu TyrArg 355 360 365 Arg Pro Phe Asn Ile Gly Ile Asn Asn Gln Gln Leu Ser ValLeu Asp 370 375 380 Gly Thr Glu Phe Ala Tyr Gly Thr Ser Ser Asn Leu ProSer Ala Val 385 390 395 400 Tyr Arg Lys Ser Gly Thr Val Asp Ser Leu AspGlu Ile Pro Pro Gln 405 410 415 Asn Asn Asn Val Pro Pro Arg Gln Gly PheSer His Arg Leu Ser His 420 425 430 Val Ser Met Phe Arg Ser Gly Phe SerAsn Ser Ser Val Ser Ile Ile 435 440 445 Arg Ala Pro Met Phe Ser Trp IleHis Arg Ser Ala Glu Phe Asn Asn 450 455 460 Ile Ile Ala Ser Asp Ser IleThr Gln Ile Pro Ala Val Lys Gly Asn 465 470 475 480 Phe Leu Phe Asn GlySer Val Ile Ser Gly Pro Gly Phe Thr Gly Gly 485 490 495 Asp Leu Val ArgLeu Asn Ser Ser Gly Asn Asn Ile Gln Asn Arg Gly 500 505 510 Tyr Ile GluVal Pro Ile His Phe Pro Ser Thr Ser Thr Arg Tyr Arg 515 520 525 Val ArgVal Arg Tyr Ala Ser Val Thr Pro Ile His Leu Asn Val Asn 530 535 540 TrpGly Asn Ser Ser Ile Phe Ser Asn Thr Val Pro Ala Thr Ala Thr 545 550 555560 Ser Leu Asp Asn Leu Gln Ser Ser Asp Phe Gly Tyr Phe Glu Ser Ala 565570 575 Asn Ala Phe Thr Ser Ser Leu Gly Asn Ile Val Gly Val Arg Asn Phe580 585 590 Ser Gly Thr Ala Gly Val Ile Ile Asp Arg Phe Glu Phe Ile ProVal 595 600 605 Thr Ala Thr Leu Glu Ala Glu Tyr Asn Leu Glu Arg Ala GlnLys Ala 610 615 620 Val Asn Ala Leu Phe Thr Ser Thr Asn Gln Leu Gly LeuLys Thr Asn 625 630 635 640 Val Thr Asp Tyr His Ile Asp Gln Val Ser AsnLeu Val Thr Tyr Leu 645 650 655 Ser Asp Glu Phe Cys Leu Asp Glu Lys ArgGlu Leu Ser Glu Lys Val 660 665 670 Lys His Ala Lys Arg Leu Ser Asp GluArg Asn Leu Leu Gln Asp Ser 675 680 685 Asn Phe Lys Asp Ile Asn Arg GlnPro Glu Arg Gly Trp Gly Gly Ser 690 695 700 Thr Gly Ile Thr Ile Gln GlyGly Asp Asp Val Phe Lys Glu Asn Tyr 705 710 715 720 Val Thr Leu Ser GlyThr Phe Asp Glu Cys Tyr Pro Thr Tyr Leu Tyr 725 730 735 Gln Lys Ile AspGlu Ser Lys Leu Lys Ala Phe Thr Arg Tyr Gln Leu 740 745 750 Arg Gly TyrIle Glu Asp Ser Gln Asp Leu Glu Ile Tyr Leu Ile Arg 755 760 765 Tyr AsnAla Lys His Glu Thr Val Asn Val Pro Gly Thr Gly Ser Leu 770 775 780 TrpPro Leu Ser Ala Gln Ser Pro Ile Gly Lys Cys Gly Glu Pro Asn 785 790 795800 Arg Cys Ala Pro His Leu Glu Trp Asn Pro Asp Leu Asp Cys Ser Cys 805810 815 Arg Asp Gly Glu Lys Cys Ala His His Ser His His Phe Ser Leu Asp820 825 830 Ile Asp Val Gly Cys Thr Asp Leu Asn Glu Asp Leu Gly Val TrpVal 835 840 845 Ile Phe Lys Ile Lys Thr Gln Asp Gly His Ala Arg Leu GlyAsn Leu 850 855 860 Glu Phe Leu Glu Glu Lys Pro Leu Val Gly Glu Ala LeuAla Arg Val 865 870 875 880 Lys Arg Ala Glu Lys Lys Trp Arg Asp Lys ArgGlu Lys Leu Glu Trp 885 890 895 Glu Thr Asn Ile Val Tyr Lys Glu Ala LysGlu Ser Val Asp Ala Leu 900 905 910 Phe Val Asn Ser Gln Tyr Asp Gln LeuGln Ala Asp Thr Asn Ile Ala 915 920 925 Met Ile His Ala Ala Asp Lys ArgVal His Ser Ile Arg Glu Ala Tyr 930 935 940 Leu Pro Glu Leu Ser Val IlePro Gly Val Asn Ala Ala Ile Phe Glu 945 950 955 960 Glu Leu Glu Gly ArgIle Phe Thr Ala Phe Ser Leu Tyr Asp Ala Arg 965 970 975 Asn Val Ile LysAsn Gly Asp Phe Asn Asn Gly Leu Ser Cys Trp Asn 980 985 990 Val Lys GlyHis Val Asp Val Glu Glu Gln Asn Asn Gln Arg Ser Val 995 1000 1005 LeuVal Val Pro Glu Trp Glu Ala Glu Val Ser Gln Glu Val Arg Val 1010 10151020 Cys Pro Gly Arg Gly Tyr Ile Leu Arg Val Thr Ala Tyr Lys Glu Gly1025 1030 1035 1040 Tyr Gly Glu Gly Cys Val Thr Ile His Glu Ile Glu AsnAsn Thr Asp 1045 1050 1055 Glu Leu Lys Phe Ser Asn Cys Val Glu Glu GluIle Tyr Pro Asn Asn 1060 1065 1070 Thr Val Thr Cys Asn Asp Tyr Thr ValAsn Gln Glu Glu Tyr Gly Gly 1075 1080 1085 Ala Tyr Thr Ser Arg Asn ArgGly Tyr Asn Glu Ala Pro Ser Val Pro 1090 1095 1100 Ala Asp Tyr Ala SerVal Tyr Glu Glu Lys Ser Tyr Thr Asp Gly Arg 1105 1110 1115 1120 Arg GluAsn Pro Cys Glu Phe Asn Arg Gly Tyr Arg Asp Tyr Thr Pro 1125 1130 1135Leu Pro Val Gly Tyr Val Thr Lys Glu Leu Glu Tyr Phe Pro Glu Thr 11401145 1150 Asp Lys Val Trp Ile Glu Ile Gly Glu Thr Glu Gly Thr Phe IleVal 1155 1160 1165 Asp Ser Val Glu Leu Leu Leu Met Glu Glu 1170 1175 7719 PRT Bacillus thuringiensis 7 Met Lys Leu Lys Asn Gln Asp Lys His GlnSer Phe Ser Ser Asn Ala 1 5 10 15 Lys Val Asp Lys Ile Ser Thr Asp SerLeu Lys Asn Glu Thr Asp Ile 20 25 30 Glu Leu Gln Asn Ile Asn His Glu AspCys Leu Lys Met Ser Glu Tyr 35 40 45 Glu Asn Val Glu Pro Phe Val Ser AlaSer Thr Ile Gln Thr Gly Ile 50 55 60 Gly Ile Ala Gly Lys Ile Leu Gly ThrLeu Gly Val Pro Phe Ala Gly 65 70 75 80 Gln Val Ala Ser Leu Tyr Ser PheIle Leu Gly Glu Leu Trp Pro Lys 85 90 95 Gly Lys Asn Gln Trp Glu Ile PheMet Glu His Val Glu Glu Ile Ile 100 105 110 Asn Gln Lys Ile Ser Thr TyrAla Arg Asn Lys Ala Leu Thr Asp Leu 115 120 125 Lys Gly Leu Gly Asp AlaLeu Ala Val Tyr His Asp Ser Leu Glu Ser 130 135 140 Trp Val Gly Asn ArgAsn Asn Thr Arg Ala Arg Ser Val Val Lys Ser 145 150 155 160 Gln Tyr IleAla Leu Glu Leu Met Phe Val Gln Lys Leu Pro Ser Phe 165 170 175 Ala ValSer Gly Glu Glu Val Pro Leu Leu Pro Ile Tyr Ala Gln Ala 180 185 190 AlaAsn Leu His Leu Leu Leu Leu Arg Asp Ala Ser Ile Phe Gly Lys 195 200 205Glu Trp Gly Leu Ser Ser Ser Glu Ile Ser Thr Phe Tyr Asn Arg Gln 210 215220 Val Glu Arg Ala Gly Asp Tyr Ser Asp His Cys Val Lys Trp Tyr Ser 225230 235 240 Thr Gly Leu Asn Asn Leu Arg Gly Thr Asn Ala Glu Ser Trp ValArg 245 250 255 Tyr Asn Gln Phe Arg Arg Asp Met Thr Leu Met Val Leu AspLeu Val 260 265 270 Ala Leu Phe Pro Ser Tyr Asp Thr Gln Met Tyr Pro IleLys Thr Thr 275 280 285 Ala Gln Leu Thr Arg Glu Val Tyr Thr Asp Ala IleGly Thr Val His 290 295 300 Pro His Pro Ser Phe Thr Ser Thr Thr Trp TyrAsn Asn Asn Ala Pro 305 310 315 320 Ser Phe Ser Ala Ile Glu Ala Ala ValVal Arg Asn Pro His Leu Leu 325 330 335 Asp Phe Leu Glu Gln Val Thr IleTyr Ser Leu Leu Ser Arg Trp Ser 340 345 350 Asn Thr Gln Tyr Met Asn MetTrp Gly Gly His Lys Leu Glu Phe Arg 355 360 365 Thr Ile Gly Gly Thr LeuAsn Ile Ser Thr Gln Gly Ser Thr Asn Thr 370 375 380 Ser Ile Asn Pro ValThr Leu Pro Phe Thr Ser Arg Asp Val Tyr Arg 385 390 395 400 Thr Glu SerLeu Ala Gly Leu Asn Leu Phe Leu Thr Gln Pro Val Asn 405 410 415 Gly ValPro Arg Val Asp Phe His Trp Lys Phe Val Thr His Pro Ile 420 425 430 AlaSer Asp Asn Phe Tyr Tyr Pro Gly Tyr Ala Gly Ile Gly Thr Gln 435 440 445Leu Gln Asp Ser Glu Asn Glu Leu Pro Pro Glu Ala Thr Gly Gln Pro 450 455460 Asn Tyr Glu Ser Tyr Ser His Arg Leu Ser His Ile Gly Leu Ile Ser 465470 475 480 Ala Ser His Val Lys Ala Leu Val Tyr Ser Trp Thr His Arg SerAla 485 490 495 Asp Arg Thr Asn Thr Ile Glu Pro Asn Ser Ile Thr Gln IlePro Leu 500 505 510 Val Lys Ala Phe Asn Leu Ser Ser Gly Ala Ala Val ValArg Gly Pro 515 520 525 Gly Phe Thr Gly Gly Asp Ile Leu Arg Arg Thr AsnThr Gly Thr Phe 530 535 540 Gly Asp Ile Arg Val Asn Ile Asn Pro Pro PheAla Gln Arg Tyr Arg 545 550 555 560 Val Arg Ile Arg Tyr Ala Ser Thr ThrAsp Leu Gln Phe His Thr Ser 565 570 575 Ile Asn Gly Lys Ala Ile Asn GlnGly Asn Phe Ser Ala Thr Met Asn 580 585 590 Arg Gly Glu Asp Leu Asp TyrLys Thr Phe Arg Thr Val Gly Phe Thr 595 600 605 Thr Pro Phe Ser Phe LeuAsp Val Gln Ser Thr Phe Thr Ile Gly Ala 610 615 620 Trp Asn Phe Ser SerGly Asn Glu Val Tyr Ile Asp Arg Ile Glu Phe 625 630 635 640 Val Pro ValGlu Val Thr Tyr Glu Ala Glu Tyr Asp Phe Glu Lys Ala 645 650 655 Gln GluLys Val Thr Ala Leu Phe Thr Ser Thr Asn Pro Arg Gly Leu 660 665 670 LysThr Asp Val Lys Asp Tyr His Ile Asp Gln Val Ser Asn Leu Val 675 680 685Glu Ser Leu Ser Asp Glu Phe Tyr Leu Asp Glu Lys Arg Glu Leu Phe 690 695700 Glu Ile Val Lys Tyr Ala Lys Gln Leu His Ile Glu Arg Asn Met 705 710715 8 652 PRT Bacillus thuringiensis 8 Met Ile Arg Lys Gly Gly Arg LysMet Asn Pro Asn Asn Arg Ser Glu 1 5 10 15 His Asp Thr Ile Lys Thr ThrGlu Asn Asn Glu Val Pro Thr Asn His 20 25 30 Val Gln Tyr Pro Leu Ala GluThr Pro Asn Pro Thr Leu Glu Asp Leu 35 40 45 Asn Tyr Lys Glu Phe Leu ArgMet Thr Ala Asp Asn Asn Thr Glu Ala 50 55 60 Leu Asp Ser Ser Thr Thr LysAsp Val Ile Gln Lys Gly Ile Ser Val 65 70 75 80 Val Gly Asp Leu Leu GlyVal Val Gly Phe Pro Phe Gly Gly Ala Leu 85 90 95 Val Ser Phe Tyr Thr AsnPhe Leu Asn Thr Ile Trp Pro Ser Glu Asp 100 105 110 Pro Trp Lys Ala PheMet Glu Gln Val Glu Ala Leu Met Asp Gln Lys 115 120 125 Ile Ala Asp TyrAla Lys Asn Lys Ala Leu Ala Glu Leu Gln Gly Leu 130 135 140 Gln Asn AsnVal Glu Asp Tyr Val Ser Ala Leu Ser Ser Trp Gln Lys 145 150 155 160 AsnPro Val Ser Ser Arg Asn Pro His Ser Gln Gly Arg Ile Arg Glu 165 170 175Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser Phe 180 185190 Ala Ile Ser Gly Tyr Glu Val Leu Phe Leu Thr Thr Tyr Ala Gln Ala 195200 205 Ala Asn Thr His Leu Phe Leu Leu Lys Asp Ala Gln Ile Tyr Gly Glu210 215 220 Glu Trp Gly Tyr Glu Lys Glu Asp Ile Ala Glu Phe Tyr Lys ArgGln 225 230 235 240 Leu Lys Leu Thr Gln Glu Tyr Thr Asp His Cys Val LysTrp Tyr Asn 245 250 255 Val Gly Leu Asp Lys Leu Arg Gly Ser Ser Tyr GluSer Trp Val Asn 260 265 270 Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu ThrVal Leu Asp Leu Ile 275 280 285 Ala Leu Phe Pro Leu Tyr Asp Val Arg LeuTyr Pro Lys Glu Val Lys 290 295 300 Thr Glu Leu Thr Arg Asp Val Leu ThrAsp Pro Ile Val Gly Val Asn 305 310 315 320 Asn Leu Arg Gly Tyr Gly ThrThr Phe Ser Asn Ile Glu Asn Tyr Ile 325 330 335 Arg Lys Pro His Leu PheAsp Tyr Leu His Arg Ile Gln Phe His Thr 340 345 350 Arg Phe Gln Pro GlyTyr Tyr Gly Asn Asp Ser Phe Asn Tyr Trp Ser 355 360 365 Gly Asn Tyr ValSer Thr Arg Pro Ser Ile Gly Ser Asn Asp Ile Ile 370 375 380 Thr Ser ProPhe Tyr Gly Asn Lys Ser Ser Glu Pro Val Gln Asn Leu 385 390 395 400 GluPhe Asn Gly Glu Lys Val Tyr Arg Ala Val Ala Asn Thr Asn Leu 405 410 415Ala Val Trp Pro Ser Ala Val Tyr Ser Gly Val Thr Lys Val Glu Phe 420 425430 Ser Gln Tyr Asn Asp Gln Thr Asp Glu Ala Ser Thr Gln Thr Tyr Asp 435440 445 Ser Lys Arg Asn Val Gly Ala Val Ser Trp Asp Ser Ile Asp Gln Leu450 455 460 Pro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Gly Tyr Ser HisGln 465 470 475 480 Leu Asn Tyr Val Met Cys Phe Leu Met Gln Gly Ser ArgGly Thr Ile 485 490 495 Pro Val Leu Thr Trp Thr His Lys Ser Val Asp PhePhe Asn Met Ile 500 505 510 Asp Ser Lys Lys Ile Thr Gln Leu Pro Leu ValLys Ala Tyr Lys Leu 515 520 525 Gln Ser Gly Ala Ser Val Val Ala Gly ProArg Phe Thr Gly Gly Asp 530 535 540 Ile Ile Gln Cys Thr Glu Asn Gly SerAla Ala Thr Ile Tyr Val Thr 545 550 555 560 Pro Asp Val Ser Tyr Ser GlnLys Tyr Arg Ala Arg Ile His Tyr Ala 565 570 575 Ser Thr Ser Gln Ile ThrPhe Thr Leu Ser Leu Asp Gly Ala Pro Phe 580 585 590 Asn Gln Tyr Tyr PheAsp Lys Thr Ile Asn Lys Gly Asp Thr Leu Thr 595 600 605 Tyr Asn Ser PheAsn Leu Ala Ser Phe Ser Thr Pro Phe Glu Leu Ser 610 615 620 Gly Asn AsnLeu Gln Ile Gly Val Thr Gly Leu Ser Ala Gly Asp Lys 625 630 635 640 ValTyr Ile Asp Lys Ile Glu Phe Ile Pro Val Asn 645 650 9 659 PRT Bacillusthuringiensis 9 Met Ile Arg Met Gly Gly Arg Lys Met Asn Pro Asn Asn ArgSer Glu 1 5 10 15 Tyr Asp Thr Ile Lys Val Thr Pro Asn Ser Glu Leu ProThr Asn His 20 25 30 Asn Gln Tyr Pro Leu Ala Asp Asn Pro Asn Ser Thr LeuGlu Glu Leu 35 40 45 Asn Tyr Lys Glu Phe Leu Arg Met Thr Ala Asp Asn SerThr Glu Val 50 55 60 Leu Asp Ser Ser Thr Val Lys Asp Ala Val Gly Thr GlyIle Ser Val 65 70 75 80 Val Gly Gln Ile Leu Gly Val Val Gly Val Pro PheAla Gly Ala Leu 85 90 95 Thr Ser Phe Tyr Gln Ser Phe Leu Asn Ala Ile TrpPro Ser Asp Ala 100 105 110 Asp Pro Trp Lys Ala Phe Met Ala Gln Val GluVal Leu Ile Asp Lys 115 120 125 Lys Ile Glu Glu Tyr Ala Lys Ser Lys AlaLeu Ala Glu Leu Gln Gly 130 135 140 Leu Gln Asn Asn Phe Glu Asp Tyr ValAsn Ala Leu Asp Ser Trp Lys 145 150 155 160 Lys Ala Pro Val Asn Leu ArgSer Arg Arg Ser Gln Asp Arg Ile Arg 165 170 175 Glu Leu Phe Ser Gln AlaGlu Ser His Phe Arg Asn Ser Met Pro Ser 180 185 190 Phe Ala Val Ser LysPhe Glu Val Leu Phe Leu Pro Thr Tyr Ala Gln 195 200 205 Ala Ala Asn ThrHis Leu Leu Leu Leu Lys Asp Ala Gln Val Phe Gly 210 215 220 Glu Glu TrpGly Tyr Ser Ser Glu Asp Ile Ala Glu Phe Tyr Gln Arg 225 230 235 240 GlnLeu Lys Leu Thr Gln Gln Tyr Thr Asp His Cys Val Asn Trp Tyr 245 250 255Asn Val Gly Leu Asn Ser Leu Arg Gly Ser Thr Tyr Asp Ala Trp Val 260 265270 Lys Phe Asn Arg Phe Arg Arg Glu Met Thr Leu Thr Val Leu Asp Leu 275280 285 Ile Val Leu Phe Pro Phe Tyr Asp Val Arg Leu Tyr Ser Lys Gly Val290 295 300 Lys Thr Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro Ile Phe ThrLeu 305 310 315 320 Asn Ala Leu Gln Glu Tyr Gly Pro Thr Phe Ser Ser IleGlu Asn Ser 325 330 335 Ile Arg Lys Pro His Leu Phe Asp Tyr Leu Arg GlyIle Glu Phe His 340 345 350 Thr Arg Leu Arg Pro Gly Tyr Ser Gly Lys AspSer Phe Asn Tyr Trp 355 360 365 Ser Gly Asn Tyr Val Glu Thr Arg Pro SerIle Gly Ser Asn Asp Thr 370 375 380 Ile Thr Ser Pro Phe Tyr Gly Asp LysSer Ile Glu Pro Ile Gln Lys 385 390 395 400 Leu Ser Phe Asp Gly Gln LysVal Tyr Arg Thr Ile Ala Asn Thr Asp 405 410 415 Ile Ala Ala Phe Pro AspGly Lys Ile Tyr Phe Gly Val Thr Lys Val 420 425 430 Asp Phe Ser Gln TyrAsp Asp Gln Lys Asn Glu Thr Ser Thr Gln Thr 435 440 445 Tyr Asp Ser LysArg Tyr Asn Gly Tyr Leu Gly Ala Gln Asp Ser Ile 450 455 460 Asp Gln LeuPro Pro Glu Thr Thr Asp Glu Pro Leu Glu Lys Ala Tyr 465 470 475 480 SerHis Gln Leu Asn Tyr Ala Glu Cys Phe Leu Met Gln Asp Arg Arg 485 490 495Gly Thr Ile Pro Phe Phe Thr Trp Thr His Arg Ser Val Asp Phe Phe 500 505510 Asn Thr Ile Asp Ala Glu Lys Ile Thr Gln Leu Pro Val Val Lys Ala 515520 525 Tyr Ala Leu Ser Ser Gly Ala Ser Ile Ile Glu Gly Pro Gly Phe Thr530 535 540 Gly Gly Asn Leu Leu Phe Leu Lys Glu Ser Ser Asn Ser Ile AlaLys 545 550 555 560 Phe Lys Val Thr Leu Asn Ser Ala Ala Leu Leu Gln ArgTyr Arg Val 565 570 575 Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu Arg LeuPhe Val Gln Asn 580 585 590 Ser Asn Asn Asp Phe Leu Val Ile Tyr Ile AsnLys Thr Met Asn Ile 595 600 605 Asp Gly Asp Leu Thr Tyr Gln Thr Phe AspPhe Ala Thr Ser Asn Ser 610 615 620 Asn Met Gly Phe Ser Gly Asp Thr AsnAsp Phe Ile Ile Gly Ala Glu 625 630 635 640 Ser Phe Val Ser Asn Glu LysIle Tyr Ile Asp Lys Ile Glu Phe Ile 645 650 655 Pro Val Gln 10 1180 PRTBacillus thuringiensis 10 Met Asn Pro Tyr Gln Asn Lys Asn Glu Tyr GluThr Leu Asn Ala Ser 1 5 10 15 Gln Lys Lys Leu Asn Ile Ser Asn Asn TyrThr Arg Tyr Pro Ile Glu 20 25 30 Asn Ser Pro Lys Gln Leu Leu Gln Ser ThrAsn Tyr Lys Asp Trp Leu 35 40 45 Asn Met Cys Gln Gln Asn Gln Gln Tyr GlyGly Asp Phe Glu Thr Phe 50 55 60 Ile Asp Ser Gly Glu Leu Ser Ala Tyr ThrIle Val Val Gly Thr Val 65 70 75 80 Leu Thr Gly Phe Gly Phe Thr Thr ProLeu Gly Leu Ala Leu Ile Gly 85 90 95 Phe Gly Thr Leu Ile Pro Val Leu PhePro Ala Gln Asp Gln Ser Asn 100 105 110 Thr Trp Ser Asp Phe Ile Thr GlnThr Lys Asn Ile Ile Lys Lys Glu 115 120 125 Ile Ala Ser Thr Tyr Ile SerAsn Ala Asn Lys Ile Leu Asn Arg Ser 130 135 140 Phe Asn Val Ile Ser ThrTyr His Asn His Leu Lys Thr Trp Glu Asn 145 150 155 160 Asn Pro Asn ProGln Asn Thr Gln Asp Val Arg Thr Gln Ile Gln Leu 165 170 175 Val His TyrHis Phe Gln Asn Val Ile Pro Glu Leu Val Asn Ser Cys 180 185 190 Pro ProAsn Pro Ser Asp Cys Asp Tyr Tyr Asn Ile Leu Val Leu Ser 195 200 205 SerTyr Ala Gln Ala Ala Asn Leu His Leu Thr Val Leu Asn Gln Ala 210 215 220Val Lys Phe Glu Ala Tyr Leu Lys Asn Asn Arg Gln Phe Asp Tyr Leu 225 230235 240 Glu Pro Leu Pro Thr Ala Ile Asp Tyr Tyr Pro Val Leu Thr Lys Ala245 250 255 Ile Glu Asp Tyr Thr Asn Tyr Cys Val Thr Thr Tyr Lys Lys GlyLeu 260 265 270 Asn Leu Ile Lys Thr Thr Pro Asp Ser Asn Leu Asp Gly AsnIle Asn 275 280 285 Trp Asn Thr Tyr Asn Thr Tyr Arg Thr Lys Met Thr ThrAla Val Leu 290 295 300 Asp Leu Val Ala Leu Phe Pro Asn Tyr Asp Val GlyLys Tyr Pro Ile 305 310 315 320 Gly Val Gln Ser Glu Leu Thr Arg Glu IleTyr Gln Val Leu Asn Phe 325 330 335 Glu Glu Ser Pro Tyr Lys Tyr Tyr AspPhe Gln Tyr Gln Glu Asp Ser 340 345 350 Leu Thr Arg Arg Pro His Leu PheThr Trp Leu Asp Ser Leu Asn Phe 355 360 365 Tyr Glu Lys Ala Gln Thr ThrPro Asn Asn Phe Phe Thr Ser His Tyr 370 375 380 Asn Met Phe His Tyr ThrLeu Asp Asn Ile Ser Gln Lys Ser Ser Val 385 390 395 400 Phe Gly Asn HisAsn Val Thr Asp Lys Leu Lys Ser Leu Gly Leu Ala 405 410 415 Thr Asn IleTyr Ile Phe Leu Leu Asn Val Ile Ser Leu Asp Asn Lys 420 425 430 Tyr LeuAsn Asp Tyr Asn Asn Ile Ser Lys Met Asp Phe Phe Ile Thr 435 440 445 AsnGly Thr Arg Leu Leu Glu Lys Glu Leu Thr Ala Gly Ser Gly Gln 450 455 460Ile Thr Tyr Asp Val Asn Lys Asn Ile Phe Gly Leu Pro Ile Leu Lys 465 470475 480 Arg Arg Glu Asn Gln Gly Asn Pro Thr Leu Phe Pro Thr Tyr Asp Asn485 490 495 Tyr Ser His Ile Leu Ser Phe Ile Lys Ser Leu Ser Ile Pro AlaThr 500 505 510 Tyr Lys Thr Gln Val Tyr Thr Phe Ala Trp Thr His Ser SerVal Asp 515 520 525 Pro Lys Asn Thr Ile Tyr Thr His Leu Thr Thr Gln IlePro Ala Val 530 535 540 Lys Ala Asn Ser Leu Gly Thr Ala Ser Lys Val ValGln Gly Pro Gly 545 550 555 560 His Thr Gly Gly Asp Leu Ile Asp Phe LysAsp His Phe Lys Ile Thr 565 570 575 Cys Gln His Ser Asn Phe Gln Gln SerTyr Phe Ile Arg Ile Arg Tyr 580 585 590 Ala Ser Asn Gly Ser Ala Asn ThrArg Ala Val Ile Asn Leu Ser Ile 595 600 605 Pro Gly Val Ala Glu Leu GlyMet Ala Leu Asn Pro Thr Phe Ser Gly 610 615 620 Thr Asp Tyr Thr Asn LeuLys Tyr Lys Asp Phe Gln Tyr Leu Glu Phe 625 630 635 640 Ser Asn Glu ValLys Phe Ala Pro Asn Gln Asn Ile Ser Leu Val Phe 645 650 655 Asn Arg SerAsp Val Tyr Thr Asn Thr Thr Val Leu Ile Asp Lys Ile 660 665 670 Glu PheLeu Pro Ile Thr Arg Ser Ile Arg Glu Asp Arg Glu Lys Gln 675 680 685 LysLeu Glu Thr Val Gln Gln Ile Ile Asn Thr Phe Tyr Ala Asn Pro 690 695 700Ile Lys Asn Thr Leu Gln Ser Glu Leu Thr Asp Tyr Asp Ile Asp Gln 705 710715 720 Ala Ala Asn Leu Val Glu Cys Ile Ser Glu Glu Leu Tyr Pro Lys Glu725 730 735 Lys Met Leu Leu Leu Asp Glu Val Lys Asn Ala Lys Gln Leu SerGln 740 745 750 Ser Arg Asn Val Leu Gln Asn Gly Asp Phe Glu Ser Ala ThrLeu Gly 755 760 765 Trp Thr Thr Ser Asp Asn Ile Thr Ile Gln Glu Asp AspPro Ile Phe 770 775 780 Lys Gly His Tyr Leu His Met Ser Gly Ala Arg AspIle Asp Gly Thr 785 790 795 800 Ile Phe Pro Thr Tyr Ile Phe Gln Lys IleAsp Glu Ser Lys Leu Lys 805 810 815 Pro Tyr Thr Arg Tyr Leu Val Arg GlyPhe Val Gly Ser Ser Lys Asp 820 825 830 Val Glu Leu Val Val Ser Arg TyrGly Glu Glu Ile Asp Ala Ile Met 835 840 845 Asn Val Pro Ala Asp Leu AsnTyr Leu Tyr Pro Ser Thr Phe Asp Cys 850 855 860 Glu Gly Ser Asn Arg CysGlu Thr Ser Ala Val Pro Ala Asn Ile Gly 865 870 875 880 Asn Thr Ser AspMet Leu Tyr Ser Cys Gln Tyr Asp Thr Gly Lys Lys 885 890 895 His Val ValCys Gln Asp Ser His Gln Phe Ser Phe Thr Ile Asp Thr 900 905 910 Gly AlaLeu Asp Thr Asn Glu Asn Ile Gly Val Trp Val Met Phe Lys 915 920 925 IleSer Ser Pro Asp Gly Tyr Ala Ser Leu Asp Asn Leu Glu Val Ile 930 935 940Glu Glu Gly Pro Ile Asp Gly Glu Ala Leu Ser Arg Val Lys His Met 945 950955 960 Glu Lys Lys Trp Asn Asp Gln Met Glu Ala Lys Arg Ser Glu Thr Gln965 970 975 Gln Ala Tyr Asp Val Ala Lys Gln Ala Ile Asp Ala Leu Phe ThrAsn 980 985 990 Val Gln Asp Glu Ala Leu Gln Phe Asp Thr Thr Leu Ala GlnIle Gln 995 1000 1005 Tyr Ala Glu Tyr Leu Val Gln Ser Ile Pro Tyr ValTyr Asn Asp Trp 1010 1015 1020 Leu Ser Asp Val Pro Gly Met Asn Tyr AspIle Tyr Val Glu Leu Asp 1025 1030 1035 1040 Ala Arg Val Ala Gln Ala ArgTyr Leu Tyr Asp Thr Arg Asn Ile Ile 1045 1050 1055 Lys Asn Gly Asp PheThr Gln Gly Val Met Gly Trp His Val Thr Gly 1060 1065 1070 Asn Ala AspVal Gln Gln Ile Asp Gly Val Ser Val Leu Val Leu Ser 1075 1080 1085 AsnTrp Ser Ala Gly Val Ser Gln Asn Val His Leu Gln His Asn His 1090 10951100 Gly Tyr Val Leu Arg Val Ile Ala Lys Lys Glu Gly Pro Gly Asn Gly1105 1110 1115 1120 Tyr Val Thr Leu Met Asp Cys Glu Glu Asn Gln Glu LysLeu Thr Phe 1125 1130 1135 Thr Ser Cys Glu Glu Gly Tyr Ile Thr Lys ThrVal Asp Val Phe Pro 1140 1145 1150 Asp Thr Asp Arg Val Arg Ile Glu IleGly Glu Thr Glu Gly Ser Phe 1155 1160 1165 Tyr Ile Glu Ser Ile Glu LeuIle Cys Met Asn Glu 1170 1175 1180 11 475 PRT Bacillus thuringiensis 11Met Ile Ile Asp Ser Lys Thr Thr Leu Pro Arg His Ser Leu Ile His 1 5 1015 Thr Ile Lys Leu Asn Ser Asn Lys Lys Tyr Gly Pro Gly Asp Met Thr 20 2530 Asn Gly Asn Gln Phe Ile Ile Ser Lys Gln Glu Trp Ala Thr Ile Gly 35 4045 Ala Tyr Ile Gln Thr Gly Leu Gly Leu Pro Val Asn Glu Gln Gln Leu 50 5560 Arg Thr His Val Asn Leu Ser Gln Asp Ile Ser Ile Pro Ser Asp Phe 65 7075 80 Ser Gln Leu Tyr Asp Val Tyr Cys Ser Asp Lys Thr Ser Ala Glu Trp 8590 95 Trp Asn Lys Asn Leu Tyr Pro Leu Ile Ile Lys Ser Ala Asn Asp Ile100 105 110 Ala Ser Tyr Gly Phe Lys Val Ala Gly Asp Pro Ser Ile Lys LysAsp 115 120 125 Gly Tyr Phe Lys Lys Leu Gln Asp Glu Leu Asp Asn Ile ValAsp Asn 130 135 140 Asn Ser Asp Asp Asp Ala Ile Ala Lys Ala Ile Lys AspPhe Lys Ala 145 150 155 160 Arg Cys Gly Ile Leu Ile Lys Glu Ala Lys GlnTyr Glu Glu Ala Ala 165 170 175 Lys Asn Ile Val Thr Ser Leu Asp Gln PheLeu His Gly Asp Gln Lys 180 185 190 Lys Leu Glu Gly Val Ile Asn Ile GlnLys Arg Leu Lys Glu Val Gln 195 200 205 Thr Ala Leu Asn Gln Ala His GlyGlu Ser Ser Pro Ala His Lys Glu 210 215 220 Leu Leu Glu Lys Val Lys AsnLeu Lys Thr Thr Leu Glu Arg Thr Ile 225 230 235 240 Lys Ala Glu Gln AspLeu Glu Lys Lys Val Glu Tyr Ser Phe Leu Leu 245 250 255 Gly Pro Leu LeuGly Phe Val Val Tyr Glu Ile Leu Glu Asn Thr Ala 260 265 270 Val Gln HisIle Lys Asn Gln Ile Asp Glu Ile Lys Lys Gln Leu Asp 275 280 285 Ser AlaGln His Asp Leu Asp Arg Asp Val Lys Ile Ile Gly Met Leu 290 295 300 AsnSer Ile Asn Thr Asp Ile Asp Asn Leu Tyr Ser Gln Gly Gln Glu 305 310 315320 Ala Ile Lys Val Phe Gln Lys Leu Gln Gly Ile Trp Ala Thr Ile Gly 325330 335 Ala Gln Ile Glu Asn Leu Arg Thr Thr Ser Leu Gln Glu Val Gln Asp340 345 350 Ser Asp Asp Ala Asp Glu Ile Gln Ile Glu Leu Glu Asp Ala SerAsp 355 360 365 Ala Trp Leu Val Val Ala Gln Glu Ala Arg Asp Phe Thr LeuAsn Ala 370 375 380 Tyr Ser Thr Asn Ser Arg Gln Asn Leu Pro Ile Asn ValIle Ser Asp 385 390 395 400 Ser Cys Asn Cys Ser Thr Thr Asn Met Thr SerAsn Gln Tyr Ser Asn 405 410 415 Pro Thr Thr Asn Met Thr Ser Asn Gln TyrMet Ile Ser His Glu Tyr 420 425 430 Thr Ser Leu Pro Asn Asn Phe Met LeuSer Arg Asn Ser Asn Leu Glu 435 440 445 Tyr Lys Cys Pro Glu Asn Asn PheMet Ile Tyr Trp Tyr Asn Asn Ser 450 455 460 Asp Trp Tyr Asn Asn Ser AspTrp Tyr Asn Asn 465 470 475 12 1138 PRT Bacillus thuringiensis 12 MetAsn Leu Asn Asn Leu Asp Gly Tyr Glu Asp Ser Asn Arg Thr Leu 1 5 10 15Asn Asn Ser Leu Asn Tyr Pro Thr Gln Lys Ala Leu Ser Pro Ser Leu 20 25 30Lys Asn Met Asn Tyr Gln Asp Phe Leu Ser Ile Thr Glu Arg Glu Gln 35 40 45Pro Glu Ala Leu Ala Ser Gly Asn Thr Ala Ile Asn Thr Val Val Ser 50 55 60Val Thr Gly Ala Thr Leu Ser Ala Leu Gly Val Pro Gly Ala Ser Phe 65 70 7580 Ile Thr Asn Phe Tyr Leu Lys Ile Ala Gly Leu Leu Trp Pro Glu Asn 85 9095 Gly Lys Ile Trp Asp Glu Phe Met Thr Glu Val Glu Ala Leu Ile Asp 100105 110 Gln Lys Ile Glu Glu Tyr Val Arg Asn Lys Ala Ile Ala Glu Leu Asp115 120 125 Gly Leu Gly Ser Ala Leu Asp Lys Tyr Gln Lys Ala Leu Ala AspTrp 130 135 140 Leu Gly Lys Gln Asp Asp Pro Glu Ala Ile Leu Ser Val AlaThr Glu 145 150 155 160 Phe Arg Ile Ile Asp Ser Leu Phe Glu Phe Ser MetPro Ser Phe Lys 165 170 175 Val Thr Gly Tyr Glu Ile Pro Leu Leu Thr ValTyr Ala Gln Ala Ala 180 185 190 Asn Leu His Leu Ala Leu Leu Arg Asp SerThr Leu Tyr Gly Asp Lys 195 200 205 Trp Gly Phe Thr Gln Asn Asn Ile GluGlu Asn Tyr Asn Arg Gln Lys 210 215 220 Lys Arg Ile Ser Glu Tyr Ser AspHis Cys Thr Lys Trp Tyr Asn Ser 225 230 235 240 Gly Leu Ser Arg Leu AsnGly Ser Thr Tyr Glu Gln Trp Ile Asn Tyr 245 250 255 Asn Arg Phe Arg ArgGlu Met Ile Leu Met Ala Leu Asp Leu Val Ala 260 265 270 Val Phe Pro PheHis Asp Pro Arg Arg Tyr Ser Met Glu Thr Ser Thr 275 280 285 Gln Leu ThrArg Glu Val Tyr Thr Asp Pro Val Ser Leu Ser Ile Ser 290 295 300 Asn ProAsp Ile Gly Pro Ser Phe Ser Gln Met Glu Asn Thr Ala Ile 305 310 315 320Arg Thr Pro His Leu Val Asp Tyr Leu Asp Glu Leu Tyr Ile Tyr Thr 325 330335 Ser Lys Tyr Lys Ala Phe Ser His Glu Ile Gln Pro Asp Leu Phe Tyr 340345 350 Trp Ser Ala His Lys Val Ser Phe Lys Lys Ser Glu Gln Ser Asn Leu355 360 365 Tyr Thr Thr Gly Ile Tyr Gly Lys Thr Ser Gly Tyr Ile Ser SerGly 370 375 380 Ala Tyr Ser Phe His Gly Asn Asp Ile Tyr Arg Thr Leu AlaAla Pro 385 390 395 400 Ser Val Val Val Tyr Pro Tyr Thr Gln Asn Tyr GlyVal Glu Gln Val 405 410 415 Glu Phe Tyr Gly Val Lys Gly His Val His TyrArg Gly Asp Asn Lys 420 425 430 Tyr Asp Leu Thr Tyr Asp Ser Ile Asp GlnLeu Pro Pro Asp Gly Glu 435 440 445 Pro Ile His Glu Lys Tyr Thr His ArgLeu Cys His Ala Thr Ala Ile 450 455 460 Phe Lys Ser Thr Pro Asp Tyr AspAsn Ala Thr Ile Pro Ile Phe Ser 465 470 475 480 Trp Thr His Arg Ser AlaGlu Tyr Tyr Asn Arg Ile Tyr Pro Asn Lys 485 490 495 Ile Thr Lys Ile ProAla Val Lys Met Tyr Lys Leu Asp Asp Pro Ser 500 505 510 Thr Val Val LysGly Pro Gly Phe Thr Gly Gly Asp Leu Val Lys Arg 515 520 525 Gly Ser ThrGly Tyr Ile Gly Asp Ile Lys Ala Thr Val Asn Ser Pro 530 535 540 Leu SerGln Lys Tyr Arg Val Arg Val Arg Tyr Ala Thr Asn Val Ser 545 550 555 560Gly Gln Phe Asn Val Tyr Ile Asn Asp Lys Ile Thr Leu Gln Thr Lys 565 570575 Phe Gln Asn Thr Val Glu Thr Ile Gly Glu Gly Lys Asp Leu Thr Tyr 580585 590 Gly Ser Phe Gly Tyr Ile Glu Tyr Ser Thr Thr Ile Gln Phe Pro Asp595 600 605 Glu His Pro Lys Ile Thr Leu His Leu Ser Asp Leu Ser Asn AsnSer 610 615 620 Ser Phe Tyr Val Asp Ser Ile Glu Phe Ile Pro Val Asp ValAsn Tyr 625 630 635 640 Ala Glu Lys Glu Lys Leu Glu Lys Ala Gln Lys AlaVal Asn Thr Leu 645 650 655 Phe Thr Glu Gly Arg Asn Ala Leu Gln Lys AspVal Thr Asp Tyr Lys 660 665 670 Val Asp Gln Val Ser Ile Leu Val Asp CysIle Ser Gly Asp Leu Tyr 675 680 685 Pro Asn Glu Lys Arg Glu Leu Gln AsnLeu Val Lys Tyr Ala Lys Arg 690 695 700 Leu Ser Tyr Ser Arg Asn Leu LeuLeu Asp Pro Thr Phe Asp Ser Ile 705 710 715 720 Asn Ser Ser Glu Glu AsnGly Trp Tyr Gly Ser Asn Gly Ile Val Ile 725 730 735 Gly Asn Gly Asp PheVal Phe Lys Gly Asn Tyr Leu Ile Phe Ser Gly 740 745 750 Thr Asn Asp ThrGln Tyr Pro Thr Tyr Leu Tyr Gln Lys Ile Asp Glu 755 760 765 Ser Lys LeuLys Glu Tyr Thr Arg Tyr Lys Leu Lys Gly Phe Ile Glu 770 775 780 Ser SerGln Asp Leu Glu Ala Tyr Val Ile Arg Tyr Asp Ala Lys His 785 790 795 800Arg Thr Leu Asp Val Ser Asp Asn Leu Leu Pro Asp Ile Leu Pro Glu 805 810815 Asn Thr Cys Gly Glu Pro Asn Arg Cys Ala Ala Gln Gln Tyr Leu Asp 820825 830 Glu Asn Pro Ser Pro Glu Cys Ser Ser Met Gln Asp Gly Ile Leu Ser835 840 845 Asp Ser His Ser Phe Ser Leu Asn Ile Asp Thr Gly Ser Ile AsnHis 850 855 860 Asn Glu Asn Leu Gly Ile Trp Val Leu Phe Lys Ile Ser ThrLeu Glu 865 870 875 880 Gly Tyr Ala Lys Phe Gly Asn Leu Glu Val Ile GluAsp Gly Pro Val 885 890 895 Ile Gly Glu Ala Leu Ala Arg Val Lys Arg GlnGlu Thr Lys Trp Arg 900 905 910 Asn Lys Leu Ala Gln Leu Thr Thr Glu ThrGln Ala Ile Tyr Thr Arg 915 920 925 Ala Lys Gln Ala Leu Asp Asn Leu PheAla Asn Ala Gln Asp Ser His 930 935 940 Leu Lys Arg Asp Val Thr Phe AlaGlu Ile Ala Ala Ala Arg Lys Ile 945 950 955 960 Val Gln Ser Ile Arg GluAla Tyr Met Ser Trp Leu Ser Val Val Pro 965 970 975 Gly Val Asn His ProIle Phe Thr Glu Leu Ser Gly Arg Val Gln Arg 980 985 990 Ala Phe Gln LeuTyr Asp Val Arg Asn Val Val Arg Asn Gly Arg Phe 995 1000 1005 Leu AsnGly Leu Ser Asp Trp Ile Val Thr Ser Asp Val Lys Val Gln 1010 1015 1020Glu Glu Asn Gly Asn Asn Val Leu Val Leu Asn Asn Trp Asp Ala Gln 10251030 1035 1040 Val Leu Gln Asn Val Lys Leu Tyr Gln Asp Arg Gly Tyr IleLeu His 1045 1050 1055 Val Thr Ala Arg Lys Ile Gly Ile Gly Glu Gly TyrIle Thr Ile Thr 1060 1065 1070 Asp Glu Glu Gly His Thr Asp Gln Leu ArgPhe Thr Ala Cys Glu Glu 1075 1080 1085 Ile Asp Ala Ser Asn Ala Phe IleSer Gly Tyr Ile Thr Lys Glu Leu 1090 1095 1100 Glu Phe Phe Pro Asp ThrGlu Lys Val His Ile Glu Ile Gly Glu Thr 1105 1110 1115 1120 Glu Gly IlePhe Leu Val Glu Ser Ile Glu Leu Phe Leu Met Glu Glu 1125 1130 1135 LeuCys 13 1157 PRT Bacillus thuringiensis 13 Met Ser Pro Asn Asn Gln AsnGlu Tyr Glu Ile Ile Asp Ala Thr Pro 1 5 10 15 Ser Thr Ser Val Ser SerAsp Ser Asn Arg Tyr Pro Phe Ala Asn Glu 20 25 30 Pro Thr Asp Ala Leu GlnAsn Met Asn Tyr Lys Asp Tyr Leu Lys Met 35 40 45 Ser Gly Gly Glu Asn ProGlu Leu Phe Gly Asn Pro Glu Thr Phe Ile 50 55 60 Ser Ser Ser Thr Ile GlnThr Gly Ile Gly Ile Val Gly Arg Ile Leu 65 70 75 80 Gly Ala Leu Gly ValPro Phe Ala Ser Gln Ile Ala Ser Phe Tyr Ser 85 90 95 Phe Ile Val Gly GlnLeu Trp Pro Ser Lys Ser Val Asp Ile Trp Gly 100 105 110 Glu Ile Met GluArg Val Glu Glu Leu Val Asp Gln Lys Ile Glu Lys 115 120 125 Tyr Val LysAsp Lys Ala Leu Ala Glu Leu Lys Gly Leu Gly Asn Ala 130 135 140 Leu AspVal Tyr Gln Gln Ser Leu Glu Asp Trp Leu Glu Asn Arg Asn 145 150 155 160Asp Ala Arg Thr Arg Ser Val Val Ser Asn Gln Phe Ile Ala Leu Asp 165 170175 Leu Asn Phe Val Ser Ser Ile Pro Ser Phe Ala Val Ser Gly His Glu 180185 190 Val Leu Leu Leu Ala Val Tyr Ala Gln Ala Val Asn Leu His Leu Leu195 200 205 Leu Leu Arg Asp Ala Ser Ile Phe Gly Glu Glu Trp Gly Phe ThrPro 210 215 220 Gly Glu Ile Ser Arg Phe Tyr Asn Arg Gln Val Gln Leu ThrAla Glu 225 230 235 240 Tyr Ser Asp Tyr Cys Val Lys Trp Tyr Lys Ile GlyLeu Asp Lys Leu 245 250 255 Lys Gly Thr Thr Ser Lys Ser Trp Leu Asn TyrHis Gln Phe Arg Arg 260 265 270 Glu Met Thr Leu Leu Val Leu Asp Leu ValAla Leu Phe Pro Asn Tyr 275 280 285 Asp Thr His Met Tyr Pro Ile Glu ThrThr Ala Gln Leu Thr Arg Asp 290 295 300 Val Tyr Thr Asp Pro Ile Ala PheAsn Ile Val Thr Ser Thr Gly Phe 305 310 315 320 Cys Asn Pro Trp Ser ThrHis Ser Gly Ile Leu Phe Tyr Glu Val Glu 325 330 335 Asn Asn Val Ile ArgPro Pro His Leu Phe Asp Ile Leu Ser Ser Val 340 345 350 Glu Ile Asn ThrSer Arg Gly Gly Ile Thr Leu Asn Asn Asp Ala Tyr 355 360 365 Ile Asn TyrTrp Ser Gly His Thr Leu Lys Tyr Arg Arg Thr Ala Asp 370 375 380 Ser ThrVal Thr Tyr Thr Ala Asn Tyr Gly Arg Ile Thr Ser Glu Lys 385 390 395 400Asn Ser Phe Ala Leu Glu Asp Arg Asp Ile Phe Glu Ile Asn Ser Thr 405 410415 Val Ala Asn Leu Ala Asn Tyr Tyr Gln Lys Ala Tyr Gly Val Pro Gly 420425 430 Ser Trp Phe His Met Val Lys Arg Gly Thr Ser Ser Thr Thr Ala Tyr435 440 445 Leu Tyr Ser Lys Thr His Thr Ala Leu Gln Gly Cys Thr Gln ValTyr 450 455 460 Glu Ser Ser Asp Glu Ile Pro Leu Asp Arg Thr Val Pro ValAla Glu 465 470 475 480 Ser Tyr Ser His Arg Leu Ser His Ile Thr Ser HisSer Phe Ser Lys 485 490 495 Asn Gly Ser Ala Tyr Tyr Gly Ser Phe Pro ValPhe Val Trp Thr His 500 505 510 Thr Ser Ala Asp Leu Asn Asn Thr Ile TyrSer Asp Lys Ile Thr Gln 515 520 525 Ile Pro Ala Val Lys Gly Asp Met LeuTyr Leu Gly Gly Ser Val Val 530 535 540 Gln Gly Pro Gly Phe Thr Gly GlyAsp Ile Leu Lys Arg Thr Asn Pro 545 550 555 560 Ser Ile Leu Gly Thr PheAla Val Thr Val Asn Gly Ser Leu Ser Gln 565 570 575 Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr Asp Phe Glu Phe 580 585 590 Thr Leu Tyr LeuGly Asp Thr Ile Glu Lys Asn Arg Phe Asn Lys Thr 595 600 605 Met Asp AsnGly Ala Ser Leu Thr Tyr Glu Thr Phe Lys Phe Ala Ser 610 615 620 Phe IleThr Asp Phe Gln Phe Arg Glu Thr Gln Asp Lys Ile Leu Leu 625 630 635 640Ser Met Gly Asp Phe Ser Ser Gly Gln Glu Val Tyr Ile Asp Arg Ile 645 650655 Glu Phe Ile Pro Val Asp Glu Thr Tyr Glu Ala Glu Gln Asp Leu Glu 660665 670 Ala Ala Lys Lys Ala Val Asn Ala Leu Phe Thr Asn Thr Lys Asp Gly675 680 685 Leu Arg Pro Gly Val Thr Asp Tyr Glu Val Asn Gln Ala Ala AsnLeu 690 695 700 Val Glu Cys Leu Ser Asp Asp Leu Tyr Pro Asn Glu Lys ArgLeu Leu 705 710 715 720 Phe Asp Ala Val Arg Glu Ala Lys Arg Leu Ser GlyAla Arg Asn Leu 725 730 735 Leu Gln Asp Pro Asp Phe Gln Glu Ile Asn GlyGlu Asn Gly Trp Ala 740 745 750 Ala Ser Thr Gly Ile Glu Ile Val Glu GlyAsp Ala Val Phe Lys Gly 755 760 765 Arg Tyr Leu Arg Leu Pro Gly Ala ArgGlu Ile Asp Thr Glu Thr Tyr 770 775 780 Pro Thr Tyr Leu Tyr Gln Lys ValGlu Glu Gly Val Leu Lys Pro Tyr 785 790 795 800 Thr Arg Tyr Arg Leu ArgGly Phe Val Gly Ser Ser Gln Gly Leu Glu 805 810 815 Ile Tyr Thr Ile ArgHis Gln Thr Asn Arg Ile Val Lys Asn Val Pro 820 825 830 Asp Asp Leu LeuPro Asp Val Ser Pro Val Asn Ser Asp Gly Ser Ile 835 840 845 Asn Arg CysSer Glu Gln Lys Tyr Val Asn Ser Arg Leu Glu Gly Glu 850 855 860 Asn ArgSer Gly Asp Ala His Glu Phe Ser Leu Pro Ile Asp Ile Gly 865 870 875 880Glu Leu Asp Tyr Asn Glu Asn Ala Gly Ile Trp Val Gly Phe Lys Ile 885 890895 Thr Asp Pro Glu Gly Tyr Ala Thr Leu Gly Asn Leu Glu Leu Val Glu 900905 910 Glu Gly Pro Leu Ser Gly Asp Ala Leu Glu Arg Leu Gln Arg Glu Glu915 920 925 Gln Gln Trp Lys Ile Gln Met Thr Arg Arg Arg Glu Glu Thr AspArg 930 935 940 Arg Tyr Met Ala Ser Lys Gln Ala Val Asp Arg Leu Tyr AlaAsp Tyr 945 950 955 960 Gln Asp Gln Gln Leu Asn Pro Asp Val Glu Ile ThrAsp Leu Thr Ala 965 970 975 Ala Gln Asp Leu Ile Gln Ser Ile Pro Tyr ValTyr Asn Glu Met Phe 980 985 990 Pro Glu Ile Pro Gly Met Asn Tyr Thr LysPhe Thr Glu Leu Thr Asp 995 1000 1005 Arg Leu Gln Gln Ala Trp Asn LeuTyr Asp Gln Arg Asn Ala Ile Pro 1010 1015 1020 Asn Gly Asp Phe Arg AsnGly Leu Ser Asn Trp Asn Ala Thr Pro Gly 1025 1030 1035 1040 Val Glu ValGln Gln Ile Asn His Thr Ser Val Leu Val Ile Pro Asn 1045 1050 1055 TrpAsp Glu Gln Val Ser Gln Gln Phe Thr Val Gln Pro Asn Gln Arg 1060 10651070 Tyr Val Leu Arg Val Thr Ala Arg Lys Glu Gly Val Gly Asn Gly Tyr1075 1080 1085 Val Ser Ile Arg Asp Gly Gly Asn Gln Ser Glu Thr Leu ThrPhe Ser 1090 1095 1100 Ala Ser Asp Tyr Asp Thr Asn Gly Val Tyr Asn AspGln Thr Gly Tyr 1105 1110 1115 1120 Ile Thr Lys Thr Val Thr Phe Ile ProTyr Thr Asp Gln Met Trp Ile 1125 1130 1135 Glu Ile Ser Glu Thr Glu GlyThr Phe Tyr Ile Glu Ser Val Glu Leu 1140 1145 1150 Ile Val Asp Val Glu1155 14 675 PRT Bacillus thuringiensis 14 Met Asn Pro Tyr Gln Asn LysAsn Glu Tyr Glu Ile Phe Asn Ala Pro 1 5 10 15 Ser Asn Gly Phe Ser LysSer Asn Asn Tyr Ser Arg Tyr Pro Leu Ala 20 25 30 Asn Lys Pro Asn Gln ProLeu Lys Asn Thr Asn Tyr Lys Asp Trp Leu 35 40 45 Asn Val Cys Gln Asp AsnGln Gln Tyr Gly Asn Asn Ala Gly Asn Phe 50 55 60 Ala Ser Ser Glu Thr IleVal Gly Val Ser Ala Gly Ile Ile Val Val 65 70 75 80 Gly Thr Met Leu GlyAla Phe Ala Ala Pro Val Leu Ala Ala Gly Ile 85 90 95 Ile Ser Phe Gly ThrLeu Leu Pro Ile Phe Trp Gln Gly Ser Asp Pro 100 105 110 Ala Asn Val TrpGln Asp Leu Leu Asn Ile Gly Gly Arg Pro Ile Gln 115 120 125 Glu Ile AspLys Asn Ile Ile Asn Val Leu Thr Ser Ile Val Thr Pro 130 135 140 Ile LysAsn Gln Leu Asp Lys Tyr Gln Glu Phe Phe Asp Lys Trp Glu 145 150 155 160Pro Ala Arg Thr His Ala Asn Ala Lys Ala Val His Asp Leu Phe Thr 165 170175 Thr Leu Glu Pro Ile Ile Asp Lys Asp Leu Asp Met Leu Lys Asn Asn 180185 190 Ala Ser Tyr Arg Ile Pro Thr Leu Pro Ala Tyr Ala Gln Ile Ala Thr195 200 205 Trp His Leu Asn Leu Leu Lys His Ala Ala Thr Tyr Tyr Asn IleTrp 210 215 220 Leu Gln Asn Gln Gly Ile Asn Pro Ser Thr Phe Asn Ser SerAsn Tyr 225 230 235 240 Tyr Gln Gly Tyr Leu Lys Arg Lys Ile Gln Glu TyrThr Asp Tyr Cys 245 250 255 Ile Gln Thr Tyr Asn Ala Gly Leu Thr Met IleArg Thr Asn Thr Asn 260 265 270 Ala Thr Trp Asn Met Tyr Asn Thr Tyr ArgLeu Glu Met Thr Leu Thr 275 280 285 Val Leu Asp Leu Ile Ala Ile Phe ProAsn Tyr Asp Pro Glu Lys Tyr 290 295 300 Pro Ile Gly Val Lys Ser Glu LeuIle Arg Glu Val Tyr Thr Asn Val 305 310 315 320 Asn Ser Asp Thr Phe ArgThr Ile Thr Glu Leu Glu Asn Gly Leu Thr 325 330 335 Arg Asn Pro Thr LeuPhe Thr Trp Ile Asn Gln Gly Arg Phe Tyr Thr 340 345 350 Arg Asn Ser ArgAsp Ile Leu Asp Pro Tyr Asp Ile Phe Ser Phe Thr 355 360 365 Gly Asn GlnMet Ala Phe Thr His Thr Asn Asp Asp Arg Asn Ile Ile 370 375 380 Trp GlyAla Val His Gly Asn Ile Ile Ser Gln Asp Thr Ser Lys Val 385 390 395 400Phe Pro Phe Tyr Arg Asn Lys Pro Ile Asp Lys Val Glu Ile Val Arg 405 410415 His Arg Glu Tyr Ser Asp Ile Ile Tyr Glu Met Ile Phe Phe Ser Asn 420425 430 Ser Ser Glu Val Phe Arg Tyr Ser Ser Asn Ser Thr Ile Glu Asn Asn435 440 445 Tyr Lys Arg Thr Asp Ser Tyr Met Ile Pro Lys Gln Thr Trp LysAsn 450 455 460 Glu Glu Tyr Gly His Thr Leu Ser Tyr Ile Lys Thr Asp AsnTyr Ile 465 470 475 480 Phe Ser Val Val Arg Glu Arg Arg Arg Val Ala PheSer Trp Thr His 485 490 495 Thr Ser Val Asp Phe Gln Asn Thr Ile Asp LeuAsp Asn Ile Thr Gln 500 505 510 Ile His Ala Leu Lys Ala Leu Lys Val SerSer Asp Ser Lys Ile Val 515 520 525 Lys Gly Pro Gly His Thr Gly Gly AspLeu Val Ile Leu Lys Asp Ser 530 535 540 Met Asp Phe Arg Val Arg Phe LeuLys Asn Val Ser Arg Gln Tyr Gln 545 550 555 560 Val Arg Ile Arg Tyr AlaThr Asn Ala Pro Lys Thr Thr Val Phe Leu 565 570 575 Thr Gly Ile Asp ThrIle Ser Val Glu Leu Pro Ser Thr Thr Ser Arg 580 585 590 Gln Asn Pro AsnAla Thr Asp Leu Thr Tyr Ala Asp Phe Gly Tyr Val 595 600 605 Thr Phe ProArg Thr Val Pro Asn Lys Thr Phe Glu Gly Glu Asp Thr 610 615 620 Leu LeuMet Thr Leu Tyr Gly Thr Pro Asn His Ser Tyr Asn Ile Tyr 625 630 635 640Ile Asp Lys Ile Glu Phe Ile Pro Ile Thr Gln Ser Val Leu Asp Tyr 645 650655 Thr Glu Lys Gln Asn Ile Glu Lys Thr Gln Lys Ile Val Asn Asp Leu 660665 670 Phe Val Asn 675 15 648 PRT Bacillus thuringiensis 15 Met His TyrTyr Gly Asn Arg Asn Glu Tyr Asp Ile Leu Asn Ala Ser 1 5 10 15 Ser AsnAsp Ser Asn Met Ser Asn Thr Tyr Pro Arg Tyr Pro Leu Ala 20 25 30 Asn ProGln Gln Asp Leu Met Gln Asn Thr Asn Tyr Lys Asp Trp Leu 35 40 45 Asn ValCys Glu Gly Tyr His Ile Glu Asn Pro Arg Glu Ala Ser Val 50 55 60 Arg AlaGly Leu Gly Lys Gly Leu Gly Ile Val Ser Thr Ile Val Gly 65 70 75 80 PhePhe Gly Gly Ser Ile Ile Leu Asp Thr Ile Gly Leu Phe Tyr Gln 85 90 95 IleSer Glu Leu Leu Trp Pro Glu Asp Asp Thr Gln Gln Tyr Thr Trp 100 105 110Gln Asp Ile Met Asn His Val Glu Asp Leu Ile Asp Lys Arg Ile Thr 115 120125 Glu Val Ile Arg Gly Asn Ala Ile Arg Thr Leu Ala Asp Leu Gln Gly 130135 140 Lys Val Asp Asp Tyr Asn Asn Trp Leu Lys Lys Trp Lys Asp Asp Pro145 150 155 160 Lys Ser Thr Gly Asn Leu Ser Thr Leu Val Thr Lys Phe ThrAla Leu 165 170 175 Asp Ser Asp Phe Asn Gly Ala Ile Arg Thr Val Asn AsnGln Gly Ser 180 185 190 Pro Gly Tyr Glu Leu Leu Leu Leu Pro Val Tyr AlaGln Ile Ala Asn 195 200 205 Leu His Leu Leu Leu Leu Arg Asp Ala Gln IleTyr Gly Asp Lys Trp 210 215 220 Trp Ser Ala Arg Ala Asn Ala Arg Asp AsnTyr Tyr Gln Ile Gln Leu 225 230 235 240 Glu Lys Thr Lys Glu Tyr Thr GluTyr Cys Ile Asn Trp Tyr Asn Lys 245 250 255 Gly Leu Asn Asp Phe Arg ThrAla Gly Gln Trp Val Asn Phe Asn Arg 260 265 270 Tyr Arg Arg Glu Met ThrLeu Thr Val Leu Asp Ile Ile Ser Met Phe 275 280 285 Pro Ile Tyr Asp AlaArg Leu Tyr Pro Thr Glu Val Lys Thr Glu Leu 290 295 300 Thr Arg Glu IleTyr Ser Asp Val Ile Asn Gly Glu Ile Tyr Gly Leu 305 310 315 320 Met ThrPro Tyr Phe Ser Phe Glu Lys Ala Glu Ser Leu Tyr Thr Arg 325 330 335 AlaPro His Leu Phe Thr Trp Leu Lys Gly Phe Arg Phe Val Thr Asn 340 345 350Ser Ile Ser Tyr Trp Thr Phe Leu Ser Gly Gly Gln Asn Lys Tyr Ser 355 360365 Tyr Thr Asn Asn Ser Ser Ile Asn Glu Gly Ser Phe Arg Gly Gln Asp 370375 380 Thr Asp Tyr Gly Gly Thr Ser Ser Thr Ile Asn Ile Pro Ser Asn Ser385 390 395 400 Tyr Val Tyr Asn Leu Trp Thr Glu Asn Tyr Glu Tyr Ile TyrPro Trp 405 410 415 Gly Asp Pro Val Asn Ile Thr Lys Met Asn Phe Ser ValThr Asp Asn 420 425 430 Asn Ser Ser Lys Glu Leu Ile Tyr Gly Ala His ArgThr Asn Lys Pro 435 440 445 Val Val Arg Thr Asp Phe Asp Phe Leu Thr AsnLys Glu Gly Thr Glu 450 455 460 Leu Ala Lys Tyr Asn Asp Tyr Asn His IleLeu Ser Tyr Met Leu Ile 465 470 475 480 Asn Gly Glu Thr Phe Gly Gln LysArg His Gly Tyr Ser Phe Ala Phe 485 490 495 Thr His Ser Ser Val Asp ProAsn Asn Thr Ile Ala Ala Asn Lys Ile 500 505 510 Thr Gln Ile Pro Val ValLys Ala Ser Ser Ile Asn Gly Ser Ile Ser 515 520 525 Ile Glu Lys Gly ProGly Phe Thr Gly Gly Asp Leu Val Lys Met Arg 530 535 540 Ala Asp Ser GlyLeu Thr Met Arg Phe Lys Ala Glu Leu Leu Asp Lys 545 550 555 560 Lys TyrArg Val Arg Ile Arg Tyr Lys Cys Asn Tyr Ser Ser Lys Leu 565 570 575 IleLeu Arg Lys Trp Lys Gly Glu Gly Tyr Ile Gln Gln Gln Ile His 580 585 590Asn Ile Ser Pro Thr Tyr Gly Ala Phe Ser Tyr Leu Glu Ser Phe Thr 595 600605 Ile Thr Thr Thr Glu Asn Ile Phe Asp Leu Thr Met Glu Val Thr Tyr 610615 620 Pro Tyr Gly Arg Gln Phe Val Glu Asp Ile Pro Ser Leu Ile Leu Asp625 630 635 640 Lys Ile Glu Phe Leu Pro Thr Asn 645 16 682 PRT Bacillusthuringiensis 16 Met Asn Ser Tyr Gln Asn Lys Asn Glu Tyr Glu Ile Leu AspAla Lys 1 5 10 15 Arg Asn Thr Cys His Met Ser Asn Cys Tyr Pro Lys TyrPro Leu Ala 20 25 30 Asn Asp Pro Gln Met Tyr Leu Arg Asn Thr His Tyr LysAsp Trp Ile 35 40 45 Asn Met Cys Glu Glu Ala Ser Tyr Ala Ser Ser Gly ProSer Gln Leu 50 55 60 Phe Lys Val Gly Gly Ser Ile Val Ala Lys Ile Leu GlyMet Ile Pro 65 70 75 80 Glu Val Gly Pro Leu Leu Ser Trp Met Val Ser LeuPhe Trp Pro Thr 85 90 95 Ile Glu Glu Lys Asn Thr Val Trp Glu Asp Met IleLys Tyr Val Ala 100 105 110 Asn Leu Leu Lys Gln Glu Leu Thr Asn Asp ThrLeu Asn Arg Ala Thr 115 120 125 Ser Asn Leu Ser Gly Leu Asn Glu Ser LeuAsn Ile Tyr Asn Arg Ala 130 135 140 Leu Ala Ala Trp Lys Gln Asn Lys AsnAsn Phe Ala Ser Gly Glu Leu 145 150 155 160 Ile Arg Ser Tyr Ile Asn AspLeu His Ile Leu Phe Thr Arg Asp Ile 165 170 175 Gln Ser Asp Phe Ser LeuGly Gly Tyr Glu Thr Val Leu Leu Pro Ser 180 185 190 Tyr Ala Ser Ala AlaAsn Leu His Leu Leu Leu Leu Arg Asp Val Ala 195 200 205 Ile Tyr Gly LysGlu Leu Gly Tyr Pro Ser Thr Asp Val Glu Phe Tyr 210 215 220 Tyr Asn GluGln Lys Tyr Tyr Thr Glu Lys Tyr Ser Asn Tyr Cys Val 225 230 235 240 AsnThr Tyr Lys Ser Gly Leu Glu Ser Lys Lys Gln Ile Gly Trp Ser 245 250 255Asp Phe Asn Arg Tyr Arg Arg Glu Met Thr Leu Ser Val Leu Asp Ile 260 265270 Val Ala Leu Phe Pro Leu Tyr Asp Thr Gly Leu Tyr Pro Ser Lys Asp 275280 285 Gly Lys Ile His Val Lys Ala Glu Leu Thr Arg Glu Ile Tyr Ser Asp290 295 300 Val Ile Asn Asp His Val Tyr Gly Leu Met Val Pro Tyr Ile SerPhe 305 310 315 320 Glu His Ala Glu Ser Leu Tyr Thr Arg Arg Pro His AlaPhe Thr Trp 325 330 335 Leu Lys Gly Phe Arg Phe Val Thr Asn Ser Ile AsnSer Trp Thr Phe 340 345 350 Leu Ser Gly Gly Glu Asn Arg Tyr Phe Leu ThrHis Gly Glu Gly Thr 355 360 365 Ile Tyr Asn Gly Pro Phe Leu Gly Gln AspThr Glu Tyr Gly Gly Thr 370 375 380 Ser Ser Tyr Ile Asp Ile Ser Asn AsnSer Ser Ile Tyr Asn Leu Trp 385 390 395 400 Thr Lys Asn Tyr Glu Trp IleTyr Pro Trp Thr Asp Pro Val Asn Ile 405 410 415 Thr Lys Ile Asn Phe SerIle Thr Asp Asn Ser Asn Ser Ser Glu Ser 420 425 430 Ile Tyr Gly Ala GluArg Met Asn Lys Pro Thr Val Arg Thr Asp Phe 435 440 445 Asn Phe Leu LeuAsn Arg Ala Gly Asn Gly Pro Thr Thr Tyr Asn Asp 450 455 460 Tyr Asn HisIle Leu Ser Tyr Met Leu Ile Asn Gly Glu Thr Phe Gly 465 470 475 480 GlnLys Arg His Gly Tyr Ser Phe Ala Phe Thr His Ser Ser Val Asp 485 490 495Arg Tyr Asn Thr Ile Val Pro Asp Lys Ile Val Gln Ile Pro Ala Val 500 505510 Lys Thr Asn Leu Val Gly Ala Asn Ile Ile Lys Gly Pro Gly His Thr 515520 525 Gly Gly Asp Leu Leu Lys Leu Glu Tyr Glu Arg Phe Leu Ser Leu Arg530 535 540 Ile Lys Leu Ile Ala Ser Met Thr Phe Arg Ile Arg Ile Arg TyrAla 545 550 555 560 Ser Asn Ile Ser Gly Gln Met Met Ile Asn Ile Gly TyrGln Asn Pro 565 570 575 Thr Tyr Phe Asn Ile Ile Pro Thr Thr Ser Arg AspTyr Thr Glu Leu 580 585 590 Lys Phe Glu Asp Phe Gln Leu Val Asp Thr SerTyr Ile Tyr Ser Gly 595 600 605 Gly Pro Ser Ile Ser Ser Asn Thr Leu TrpLeu Asp Asn Phe Ser Asn 610 615 620 Gly Pro Val Ile Ile Asp Lys Ile GluPhe Ile Pro Leu Gly Ile Thr 625 630 635 640 Leu Asn Gln Ala Gln Gly TyrAsp Thr Tyr Asp Gln Asn Ala Asn Gly 645 650 655 Met Tyr His Gln Asn TyrSer Asn Ser Gly Tyr Asn Tyr Asn Gln Glu 660 665 670 Tyr Asn Thr Tyr TyrGln Ser Tyr Asn Asn 675 680 17 674 PRT Bacillus thuringiensis 17 Met AsnGln Tyr Gln Asn Lys Asn Glu Tyr Glu Ile Leu Glu Ser Ser 1 5 10 15 GlnAsn Asn Met Asn Met Pro Asn Arg Tyr Pro Phe Ala Asp Asp Pro 20 25 30 AsnAla Val Met Lys Asn Gly Asn Tyr Lys Asp Trp Val Asn Glu Cys 35 40 45 GluGly Ser Asn Ile Ser Pro Ser Pro Ala Ala Ala Ile Thr Ser Lys 50 55 60 IleVal Ser Ile Val Leu Lys Thr Leu Ala Lys Ala Val Ala Ser Ser 65 70 75 80Leu Ala Asp Ser Ile Lys Ser Ser Leu Gly Ile Ser Lys Thr Ile Thr 85 90 95Glu Asn Asn Val Ser Gln Val Ser Met Val Gln Val His Gln Ile Ile 100 105110 Asn Arg Arg Ile Gln Glu Thr Ile Leu Asp Leu Gly Glu Ser Ser Leu 115120 125 Asn Gly Leu Val Ala Ile Tyr Asn Arg Asp Tyr Leu Gly Ala Leu Glu130 135 140 Ala Trp Asn Asn Asn Lys Ser Asn Ile Asn Tyr Gln Thr Asn ValAla 145 150 155 160 Glu Ala Phe Lys Thr Val Glu Arg Glu Phe Phe Thr LysLeu Lys Gly 165 170 175 Ile Tyr Arg Thr Ser Ser Ser Gln Ile Thr Leu LeuPro Thr Phe Thr 180 185 190 Gln Ala Ala Asn Leu His Leu Ser Met Leu ArgAsp Ala Val Met Tyr 195 200 205 Gln Glu Gly Trp Asn Leu Gln Ser His IleAsn Tyr Ser Lys Glu Leu 210 215 220 Asp Asp Ala Leu Glu Asp Tyr Thr AsnTyr Cys Val Glu Val Tyr Thr 225 230 235 240 Lys Gly Leu Asn Ala Leu ArgGly Ser Thr Ala Ile Asp Trp Leu Glu 245 250 255 Phe Asn Ser Phe Arg ArgAsp Met Thr Leu Met Val Leu Asp Leu Val 260 265 270 Ala Ile Phe Pro AsnTyr Asn Pro Val Arg Tyr Pro Leu Ser Thr Lys 275 280 285 Ile Ser Leu SerArg Lys Ile Tyr Thr Asp Pro Val Gly Arg Thr Asp 290 295 300 Ser Pro SerPhe Gly Asp Trp Thr Asn Thr Gly Arg Thr Leu Ala Asn 305 310 315 320 PheAsn Asp Leu Glu Arg Glu Val Thr Asp Ser Pro Ser Leu Val Lys 325 330 335Trp Leu Gly Asp Met Thr Ile Tyr Thr Gly Ala Ile Asp Ser Tyr Arg 340 345350 Pro Thr Ser Pro Gly Asp Arg Ile Gly Val Trp Tyr Gly Asn Ile Asn 355360 365 Ala Phe Tyr His Thr Gly Arg Thr Asp Val Val Met Phe Arg Gln Thr370 375 380 Gly Asp Thr Ala Tyr Glu Asp Pro Ser Thr Phe Ile Ser Asn IleLeu 385 390 395 400 Tyr Asp Asp Ile Tyr Lys Leu Asp Leu Arg Ala Ala AlaVal Ser Thr 405 410 415 Ile Gln Gly Ala Met Asp Thr Thr Phe Gly Val SerSer Ser Arg Phe 420 425 430 Phe Asp Ile Arg Gly Arg Asn Gln Leu Tyr GlnSer Asn Lys Pro Tyr 435 440 445 Pro Ser Leu Pro Ile Thr Ile Thr Phe ProGly Glu Glu Ser Ser Glu 450 455 460 Gly Asn Ala Asn Asp Tyr Ser His LeuLeu Cys Asp Val Lys Ile Leu 465 470 475 480 Gln Glu Asp Ser Ser Asn IleCys Glu Gly Arg Ser Ser Leu Leu Ser 485 490 495 His Ala Trp Thr His AlaSer Leu Asp Arg Asn Asn Thr Ile Leu Pro 500 505 510 Asp Glu Ile Thr GlnIle Pro Ala Val Thr Ala Tyr Glu Leu Arg Gly 515 520 525 Asn Ser Ser ValVal Ala Gly Pro Gly Ser Thr Gly Gly Asp Leu Val 530 535 540 Lys Met SerTyr His Ser Val Trp Ser Phe Lys Val Tyr Cys Ser Glu 545 550 555 560 LeuLys Asn Tyr Arg Val Arg Ile Arg Tyr Ala Ser His Gly Asn Cys 565 570 575Gln Phe Leu Met Lys Arg Trp Pro Ser Thr Gly Val Ala Pro Arg Gln 580 585590 Trp Ala Arg His Asn Val Gln Gly Thr Phe Ser Asn Ser Met Arg Tyr 595600 605 Glu Ala Phe Lys Tyr Leu Asp Ile Phe Thr Ile Thr Pro Glu Glu Asn610 615 620 Asn Phe Ala Phe Thr Ile Asp Leu Glu Ser Gly Gly Asp Leu PheIle 625 630 635 640 Asp Lys Ile Glu Phe Ile Pro Val Ser Gly Ser Ala PheGlu Tyr Glu 645 650 655 Gly Lys Gln Asn Ile Glu Lys Thr Gln Lys Ala ValAsn Asp Leu Phe 660 665 670 Ile Asn

That which is claimed:
 1. An isolated nucleic acid molecule selectedfrom the group consisting of: a) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1 or 3; b) a nucleic acid moleculecomprising a nucleotide sequence having at least 95% sequence identityto the nucleotide sequence of SEQ ID NO:1 or 3, wherein said nucleotidesequence encodes a polypeptide having pesticidal activity; c) a nucleicacid molecule which encodes a polypeptide comprising the amino acidsequence of SEQ ID NO:2 or 4; d) a nucleic acid molecule comprising anucleotide sequence encoding a polypeptide having at least 95% aminoacid sequence identity to the amino acid sequence of SEQ ID NO:2 or 4,wherein said polypeptide has pesticidal activity; and, e) a complementof any of a)-d).
 2. An isolated nucleic acid molecule of claim 1,wherein said nucleotide sequence is a synthetic sequence that has beendesigned for expression in a plant.
 3. The nucleic acid molecule ofclaim 2, wherein said synthetic sequence has an increased GC content. 4.A vector comprising the nucleic acid molecule of claim
 1. 5. The vectorof claim 4, further comprising a nucleic acid molecule encoding aheterologous polypeptide.
 6. A host cell that contains the vector ofclaim
 4. 7. The host cell of claim 6 that is a bacterial host cell. 8.The host cell of claim 6 that is a plant cell.
 9. A transgenic plantcomprising the host cell of claim
 8. 10. The transgenic plant of claim9, wherein said plant is selected from the group consisting of maize,sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape.11. Transgenic seed of a plant of claim
 9. 12. An isolated polypeptideselected from the group consisting of: a) a polypeptide comprising theamino acid sequence of SEQ ID NO:2 or 4; b) a polypeptide encoded by thenucleotide sequence of SEQ ID NO:1 or 3, wherein said polypeptide haspesticidal activity; c) a polypeptide comprising an amino acid sequencehaving at least 95% sequence identity to the amino acid sequence of SEQID NO:2 or 4, wherein said polypeptide has pesticidal activity; and, d)a polypeptide that is encoded by a nucleotide sequence that is at least95% identical to the nucleotide sequence of SEQ ID NO:1 or
 3. 13. Thepolypeptide of claim 12, further comprising a heterologous amino acidsequence.
 14. An antibody that selectively binds to a polypeptide ofclaim
 12. 15. A composition comprising the polypeptide of claim
 12. 16.The composition of claim 15, wherein said composition is selected fromthe group consisting of a powder, dust, pellet, granule, spray,emulsion, colloid, and solution.
 17. The composition of claim 15,wherein said composition is prepared by desiccation, lyophilization,homogenization, extraction, filtration, centrifugation, sedimentation,or concentration of a culture of Bacillus thuringiensis cells.
 18. Thecomposition of claim 15, comprising from about 1% to about 99% by weightof said polypeptide.
 19. A method for producing a polypeptide withpesticidal activity, comprising culturing the host cell of claim 6 underconditions in which a nucleic acid molecule encoding the polypeptide isexpressed, said polypeptide being selected from the group consisting of:a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or 4;b) a polypeptide encoded by the nucleotide sequence of SEQ ID NO:1 or 3,wherein said polypeptide has pesticidal activity; c) a polypeptidecomprising an amino acid sequence having at least 95% sequence identityto the amino acid sequence of SEQ ID NO:2 or 4, wherein said polypeptidehas pesticidal activity; and, d) a polypeptide that is encoded by anucleotide sequence that is at least 95% identical to a nucleotidesequence of SEQ ID NO:1 or
 3. 20. A method for controlling alepidopteran or coleopteran pest population comprising contacting saidpopulation with a pesticidally-effective amount of a polypeptide ofclaim
 12. 21. A method for killing a lepidopteran or coleopteran pest,comprising contacting said pest with, or feeding to said pest, apesticidally-effective amount of a polypeptide of claim
 12. 22. A planthaving stably incorporated into its genome a DNA construct comprising anucleotide sequence that encodes a protein having pesticidal activity,wherein said nucleotide sequence is selected from the group consistingof: a) a nucleotide sequence of SEQ ID NO:1 or 3; b) a nucleotidesequence having at least 95% sequence identity to a nucleotide sequenceof SEQ ID NO:1 or 3, wherein said nucleotide sequence encodes apolypeptide having pesticidal activity; c) a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence of SEQ ID NO:2or 4; and, d) a nucleotide sequence encoding a polypeptide having atleast 95% amino acid sequence identity to the amino acid sequence of SEQID NO:2 or 4, wherein said polypeptide has pesticidal activity; whereinsaid nucleotide sequence is operably linked to a promoter that drivesexpression of a coding sequence in a plant cell.
 23. A plant cell havingstably incorporated into its genome a DNA construct comprising anucleotide sequence that encodes a protein having pesticidal activity,wherein said nucleotide sequence is selected from the group consistingof: a) a nucleotide sequence of SEQ ID NO:1 or 3; b) a nucleotidesequence having at least 95% sequence identity to a nucleotide sequenceof SEQ ID NO:1 or 3, wherein said nucleotide sequence encodes apolypeptide having pesticidal activity; c) a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence of SEQ ID NO:2or 4; and, d) a nucleotide sequence encoding a polypeptide having atleast 95% amino acid sequence identity to the amino acid sequence of SEQID NO:2 or 4, wherein said polypeptide has pesticidal activity; whereinsaid nucleotide sequence is operably linked to a promoter that drivesexpression of a coding sequence in a plant cell.